COMPLEMENT FACTOR Bb ANTIBODIES

ABSTRACT

The present disclosure relates to antibodies and polynucleotides encoding the same, which may be used to prevent, control, or reduce the activity of the complement pathway. In addition, the disclosure is directed to compositions and methods for diagnosing and treating diseases mediated by or involving complement Factor Bb. Specifically, the disclosure is related to anti-complement Factor Bb antibodies.

CROSS-REFERENCE

This application is a Divisional of U.S. application Ser. No.15/790,759, filed Oct. 23, 2017, now U.S. Pat. No. 10,093,724, which isa divisional of U.S. application Ser. No. 14/631,057, filed Feb. 25,2015, now U.S. Pat. No. 9,796,776, which claims priority under 35 U.S.C.§ 119(e) from U.S. Provisional Application Ser. No. 61/945,613, filedFeb. 27, 2014 and U.S. Provisional Application Ser. No. 61/947,880,filed Mar. 4, 2014, each of which is hereby incorporated by reference inits entirety.

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates to anti-complement antibodies andcompositions thereof, polynucleotides encoding the same, expressionvectors and host cells for production of the antibodies, andcompositions and methods for diagnosing and treating diseases mediatedby complement. Specifically disclosed are anti-Factor B and anti-FactorBb antibodies for use in diagnosing and treating Factor B and Factor Bbassociated diseases, in particular, Age-Related Macular Degeneration(AMD).

BACKGROUND OF THE INVENTION

The complement system is composed of nearly 50 individual proteins thatfunctions as a part of the innate immune system providing the initialphase of host defense, opsonization of foreign material, and tissuehomeostasis. (Ricklin D., 2010, Complement: a Key system for immunesurveillance and homeostasis. Nature: Immunology, 785-795) Thecomplement system is found in all multicellular organism andphylogenetically predates the formation of the adaptive immune system(Zarkadis I. K., 2001 Phylogenetic aspects of the complement system.Development and Comparative Immunology, 745-762.).

Activation of the complement system occurs along three primary pathways:classical, lectin and alternative pathways. During the activationprocess sequential protein-protein interactions and proteolytic activityleads the generation of the C3 and C5 convertases. These convertases areresponsible for producing complement activation split products thatrepresent the effector molecules of the complement cascade important foropsonization, generation of anaphylatoxins, and the formation of themembrane attack complex (MAC). The latter of these is essential for thelytic activity of the complement cascade (Ricklin D., 2010). Undernormal conditions activation of the complement cascades provides defenseagainst pathogenic bacterial, as well as clearance of diseased andinjured tissue. Normally, the formation of MAC does not affectsurrounding tissue due to the presence of cell surface and solubleregulatory components which include CFH, CFH related proteins, C4BP,CD46, CD55, CD59, and complement factor I (CFI). However, when excessactivation occurs or when there is a failure to produce complementnegative regulatory components, both acute and chronic disease statesare induced. Examples in which uncontrolled complement activation isrecognized as causative to human pathologies include:Glomerulonephritis, Systemic Lupus Erythematosus, Paroxysomal NocturnalHemoglobinuria, Alzheimer's, Hereditary Angioedmea, Myasthenia Gravisand Age-related Macular Degeneration (AMD) (Ricklin & Lambris, 2013,Complement in Immune and inflammatory Disorders: PathologicalMechanisms. Journal of Immunology, 3831-3838).

Complement factor B is a protein that circulates in the blood as asingle chain polypeptide. Upon activation of the alternative pathway,Factor B (nearly 750 aa) is cleaved by complement Factor D yielding twopolypeptides, the smaller, non-catalytic chain Ba (about 230 aa;comprising three complement control protein (CCP) domains) and thelarger, catalytic subunit Bb (about 510 aa; comprising a proteininteraction domain and a serine protease domain). Factor Bb is a serineprotease that associates with C3b to form the alternative pathway's C3convertase as well as a second protease, C5 convertase, which cleavesthe C5 protein into C5a and CSb. Cleavage product C5b initiates themembrane attack pathway, which results in the membrane attack complex(MAC). The MAC is a transmembrane channel, which results in osmoticlysis of the target pathogen. Thus, cleavage of Factor B and productionof Factor Bb aids in the complement process.

Factor B is a tightly regulated, highly specific serine protease. In itsactivated form, it catalyzes the central amplification step ofcomplement activation to initiate inflammatory responses, cell lysis,phagocytosis and B-cell stimulation (Carroll et al., Nat. Immunol.5:981-986 (2004)). Factor B is activated through an assembly process: itbinds surface-bound C3b, or its fluid-phase counterpart C3 (H₂O), afterwhich it is cleaved by factor B into fragments Ba (residues 1-234;Factor Ba, Fragment Ba, Complement Factor Ba) and Bb (residues 235-739;Factor Bb, Fragment Bb, Complement Factor Bb). Fragment Ba dissociatesfrom the complex, leaving behind the alternative pathway C3 convertasecomplex C3b-Bb, which cleaves C3 into C3a and C3b.

Age-related Macular Degeneration (AMD) is the leading cause of blindnessin the elderly in the developed nations. In the US population alone theprevalence of advanced forms of AMD are associated with vision lossoccurs in nearly 2 million individuals. Another 7 million individualswith intermediate AMD are at a high risk for development of advancedforms of AMD. Inclusion of the European population nearly doubles thenumber of impacted individuals. AMD is characterized by a progressiveloss of vision attributable to a parainflammatory process causing theprogressive degeneration of the neuroretina, and support tissues whichinclude the retinal pigmented epithelium (RPE) and choriocapillaris. Themajority of clinically significant vision loss occurs when theneurodegenerative changes impact the region of central vision within ahighly specialized region of the eye, responsible for fine visualacuity, the macula. The disease has a tremendous impact on the physicaland mental health of the individual due to vision loss and increaseddependence on family members to perform everyday tasks.

The deregulation of the complement system is highly correlated with thedevelopment of AMD. First, genetic mutations in complement genes alter aperson's risk of developing AMD. In addition, AMD-related inflammationis associated deregulation of complement activity as indicated byelevation of complement activation products in systemic circulation andin AMD tissues by histopathological analysis. New discoveries, havehighlighted the potential pathological impact of the membrane attackcomplex in disease occurrence (Whitmore S, et al. 2014, Complementactivation and choriocapillaris loss in early AMD: Implications forpathphysiology and therapy. Progress in Retinal and Eye Research, Dec.5, 2014 EPub ahead of print).

The present invention provides anti-Factor Bb antibodies for theprevention and treatment of complement associated diseases, AMD, andother complement-associated eye conditions.

SUMMARY OF THE INVENTION

The invention encompasses methods and compositions comprising ananti-Factor Bb antibody. In one embodiment, the anti-Factor Bb antibodybinds to complement Factor Bb with greater affinity than to complementFactor B. In another aspect, the anti-Factor Bb antibody binds to FactorBb and inhibits complement dependent hemolysis. In another aspect, theanti-Factor Bb antibody binds to complement Factor Bb with a Kd of lessthan about 1 nM. In another aspect, the anti-Factor Bb antibodies of theinvention blocks the formation of membrane attack complex (MAC).

In another embodiment, the anti-Factor Bb antibodies of the inventioncomprises a first amino acid sequence and a second amino acid sequencewith the first amino acid sequence being (i) a CDR1 selected from: (a) aCDR1 amino acid sequence GDIFSSHW, SEQ ID NO:1; (b) a CDR1 amino acidsequence that differs by no more than a total of 2 amino acid additions,deletions, or substitutions selected from GDIFSSHW, SEQ ID NO:1; and (c)a CDR1 amino acid sequence of GDIFSSX₁W wherein X₁ is Histidine and oneother amino acid is substituted with Alanine; (ii) a CDR2 selected from:(a) a CDR2 amino acid sequence EILPRSGITHYNENFNG, SEQ ID NO:2; (b) aCDR2 amino acid sequence that differs by no more than a total of 2 aminoacid additions, deletions, or substitutions selected fromEILPRSGITHYNENFNG, SEQ ID NO:2; and (c) a CDR2 amino acid sequence ofX₁IX₂PX₃SGITHYNENFNG wherein X₁ is Glutamic acid, X₂ is Leucine, and X₃Arginine, and one other amino acid is substituted with Alanine; and(iii) a CDR3 selected from: (a) a CDR3 amino acid sequence AINWEDS, SEQID NO:3; (b) a CDR3 amino acid sequence that differs by no more than atotal of 2 amino acid additions, deletions, or substitutions selectedfrom AINWEDS, SEQ ID NO:3; and (c) a CDR3 amino acid sequence ofAX₁NX₂X₃X₄S wherein X₁ is Isoleucine acid, X₂ is Tryptophan, X₃ Glutamicacid, X₃ Aspartic acid, and one other amino acid is substituted withAlanine; and a second amino acid sequence being (i) a CDR1 selectedfrom: (a) a CDR1 amino acid sequence HASQNVNVWL, SEQ ID NO:4; (b) a CDR1amino acid sequence that differs by no more than a total of 2 amino acidadditions, deletions, or substitutions selected from HASQNVNVWL, SEQ IDNO:4, SEQ ID NO:4; and (c) a CDR1 amino acid sequence of HASQNVNVX₁Lwherein X₁ is Tryptophan and one other amino acid is substituted withAlanine; (ii) a CDR2 selected from: (a) a CDR2 amino acid sequenceKASNLHT, SEQ ID NO:5; (b) a CDR2 amino acid sequence that differs by nomore than a total of 2 amino acid additions, deletions, or substitutionsselected from KASNLHT, SEQ ID NO:5; and (c) a CDR2 amino acid sequenceof KASNLHX₁ wherein X₁ is Threonine one other amino acid is substitutedwith Alanine; and (iii) a CDR3 selected from: (a) a CDR3 amino acidsequence QQGQSYPYT, SEQ ID NO:6; (b) a CDR3 amino acid sequence thatdiffers by no more than a total of 2 amino acid additions, deletions, orsubstitutions selected from QQGQSYPYT, SEQ ID NO:6; and (c) a CDR3 aminoacid sequence of QX₁GQSYPX₂T wherein X₁ is Glutamine acid, X₂ isTyrosine, and one other amino acid is substituted with Alanine.

In another embodiment, the anti-Factor Bb antibodies of the inventionhas a light chain variable domain amino acid sequence that is at least80% identical to a sequence selected from the group consisting of SEQ IDNO:8-11 and a heavy chain variable domain amino acid sequence that is atleast 80% identical to a sequence selected from the group consisting ofSEQ ID NO:12-15. In another embodiment, the anti-Factor Bb antibodies ofthe invention has a light chain variable domain amino acid sequence thatis at least 80% identical to a sequence selected from the groupconsisting of SEQ ID NO:24-27 and a heavy chain variable domain aminoacid sequence that is at least 80% identical to a sequence selected fromthe group consisting of SEQ ID NO:28-31. In another aspect, theanti-Factor Bb antibodies of the invention has a light chain variabledomain amino acid sequence selected from the group consisting of SEQ IDNO:8-11 and a heavy chain variable domain amino acid sequence selectedfrom the group consisting of SEQ ID NO:12-15. In another aspect, theanti-Factor Bb antibodies of the invention has a light chain variabledomain amino acid sequence that is selected from the group consisting ofSEQ ID NO:24-27 and a heavy chain variable domain amino acid sequenceselected from the group consisting of SEQ ID NO:28-31. In other aspect,the anti-Factor Bb antibody of the invention has a light chain variabledomain amino acid sequence of SEQ ID NO:11 and a heavy chain variabledomain amino acid sequence of SEQ ID NO:15.

In another embodiment, the anti-Factor Bb antibodies of the inventioncomprises a heavy chain and a light chain variable domain selected fromthe light and heavy chain variable domain amino acid sequences: SEQ IDNO:8/SEQ ID NO:12; SEQ ID NO:8/SEQ ID NO:13; SEQ ID NO:8/SEQ ID NO:14;SEQ ID NO:8/SEQ ID NO:15; SEQ ID NO:9/SEQ ID NO:12; SEQ ID NO:9/SEQ IDNO:13; SEQ ID NO:9/SEQ ID NO:14; SEQ ID NO:9/SEQ ID NO:15; SEQ IDNO:10/SEQ ID NO:12; SEQ ID NO:10/SEQ ID NO:13; SEQ ID NO:10/SEQ IDNO:14; and SEQ ID NO:10/SEQ ID NO:15; SEQ ID NO:11/SEQ ID NO:12; SEQ IDNO:11/SEQ ID NO:13; SEQ ID NO:11/SEQ ID NO:14; and SEQ ID NO:11/SEQ IDNO:15.

In another embodiment, the anti-Factor Bb antibodies of the inventioncomprises a heavy chain and a light chain variable domain selected fromthe light and heavy chain variable domain amino acid sequences: SEQ IDNO:24/SEQ ID NO:28; SEQ ID NO:24/SEQ ID NO:29; SEQ ID NO:24/SEQ IDNO:30; SEQ ID NO:24/SEQ ID NO:31; SEQ ID NO:25/SEQ ID NO:28; SEQ IDNO:25/SEQ ID NO:29; SEQ ID NO:25/SEQ ID NO:30; SEQ ID NO:25/SEQ IDNO:31; SEQ ID NO:26/SEQ ID NO:28; SEQ ID NO:26/SEQ ID NO:29; SEQ IDNO:26/SEQ ID NO:30; and SEQ ID NO:26/SEQ ID NO:31; SEQ ID NO:27/SEQ IDNO:28; SEQ ID NO:27/SEQ ID NO:29; SEQ ID NO:27/SEQ ID NO:30; and SEQ IDNO:27/SEQ ID NO:31.

In another aspect, the anti-Factor Bb antibody of the invention is amonoclonal antibody, a polyclonal antibody, a recombinant antibody, ahumanized antibody, a chimeric antibody, a multispecific antibody, or anantibody fragment. In another aspect, the anti-Factor Bb antibody of theinvention is a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fvfragment, a diabody, or a single chain antibody molecule. In anotheraspect, the anti-Factor Bb antibody of the invention is of the IgG1,IgG2, IgG3, or IgG4 type. In another aspect, the anti-Factor Bb antibodyof the invention is coupled to a labeling group. That labeling group canbe an optical label, a radioisotope, a radionuclide, an enzymatic group,or a biotinyl group.

In another embodiment, the invention is a process for preparing anisolated antibody of the invention that comprises preparing the antibodyof the invention form a host cell that secretes the antibody. In oneaspect, this means to isolate or purify the antibody from the cellculture medium in which the host cell is grown.

In another embodiment, the invention is a nucleic acid molecule encodingan isolated antibody of the invention. In one aspect, the nucleic acidmolecule encoding the antibody of the invention is operable linked to acontrol sequence.

In another embodiment, the invention is a pharmaceutical compositionthat comprises at least one antibody of the invention and apharmaceutically acceptable carrier. In one aspect, the pharmaceuticalcomposition may also comprise an additional active agent.

In another embodiment, the invention is a method for treating orpreventing a condition in a patient in need of treatment or preventioncomprising administering to said patient an effective amount of at leastone anti-Factor Bb antibody of the invention and thereby treating orpreventing the condition. In one aspect, the condition is an oculardisease. In another aspect, the condition is age-related maculardegeneration (AMD).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the binding analysis of an anti-Factor Bb monoclonalantibody, using

FIG. 2 shows the results of a hemolysis assay using either anti-Factor Bor anti-Factor Bb antibodies in the presence of 10% normal human serum.

FIG. 3 shows a diagram of the Complement Factor B structure.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification etc.Enzymatic reactions and purification techniques can be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,molecular biology, biological chemistry, physical and bio-physicalchemistry, analytical chemistry, organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsynthesis, chemical analyses, pharmaceutical preparation, formulation,and delivery and treatment of patients.

The following definitions are used herein:

“Protein,” as used herein, is meant to refer to at least two covalentlyattached amino acids, and is used interchangeably with polypeptides,oligopeptides, and peptides. The two or more covalently attached aminoacids are attached by a peptide bond.

“Factor B” refers to human Factor B, the amino acid sequence of which isshown in SEQ ID NO:16. Factor B, Protein B, Complement Factor B,Complement Protein B refer to the same sequence as SEQ ID NO:16. Otherterms can be used to refer to the same, or variants of Factor B (forexample, “preproprotein B”.) Factor Ba (SEQ ID NO:17) is one polypeptidefragment of Factor B.

“Factor Bb,” refers to a polypeptide fragment (SEQ ID NO:7) of humanFactor.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense to refer to a protein, comprising one or morepolypeptide chains that interact with a specific antigen, throughbinding of a plurality of CDRs and an epitope of the antigen. Anantibody can be a monoclonal (for e.g., full length or intact monoclonalantibodies), polyclonal, multivalent, and/or multispecific (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity). Antibodies can also be or include antibody fragments (asdescribed herein).

“Epitope” is used to refer to a sequence, structure, or molecule that isrecognized and bound by an antibody. An epitope can be referred to as an“antigenic site.”

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcR binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

“Monoclonal” antibody as used herein refers to an antibody obtained froma population of cells, wherein the population of cells isclonally-derived from a single parent cell. Monoclonal antibodies arehomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical in that they are derived from the same genesand have the same amino acid sequence and protein structure except forpossible naturally-occurring mutations that can be present in minoramounts and post-translational modifications that may, in some cases, bedifferent. Monoclonal antibodies can, in some embodiments, be highlyspecific. In some embodiments, a monoclonal antibody can be directedagainst a single antigenic site. Furthermore, in contrast to otherantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.Individual monoclonal antibodies can be produced by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present disclosure can be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or can be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or fromphage antibody libraries using the techniques described in Clackson etal. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol.222:581-597.

“Polyclonal” antibody is used to describe a heterogeneous population ofantibodies derived from a heterogeneous population of parent,antibody-producing cells. In most cases the polyclonal antibodies havedifferent affinity for differing epitopes and are produced from geneswith differing sequences.

“Chimeric” antibodies are antibodies comprising amino acid sequencesderived from two or more different species.

“Humanized” antibodies are chimeric antibodies derived from a non-humanparent antibody. In many cases specific amino acid positions in ahumanized antibody, have been changed to correspond to the identity ofthe amino acid at a corresponding position in a human antibody. In manycases, positions in a variable region of the parent (non-human) antibodyare replaced with amino acids from a variable region of a human species.This creates a humanized mouse, rat, rabbit or nonhuman-primate antibodyhaving the desired specificity, affinity, and capacity.

“Variant” refers to sequences that comprise at least one differencecompared to a parent sequence. A variant polypeptide is a protein havingat least about 75% amino acid sequence identity to a parent sequence. Avariant protein can have at least about 80% amino acid sequenceidentity, or at least about 85% amino acid sequence identity, or atleast about 90% amino acid sequence identity, or at least about 95%amino acid sequence identity, or at least about 98% amino acid sequenceidentity, or at least about 99% amino acid sequence identity with anative, or wild-type amino acid sequence. In some cases variantantibodies are antibodies having one or more difference(s) in amino acidsequence as compared to a parent antibody. Humanized and chimericantibodies are variant antibodies. Variant antibodies, therefore,comprise less than 100% sequence identity with a parent antibody.

“Isolated” or “purified” refers to a molecule that has been separatedand/or recovered from at least one component of its natural environment,wherein the component is a material that can interfere with the use, oractivity, of the molecule. Components include peptides, sugars, nucleicacids, enzymes, hormones, and other proteinaceous or nonproteinaceoussolutes.

“Complementarity Determining Regions” (CDRs) refers to one or moreregions within an antibody wherein the residues of one or more CDR aidin antigen binding. In many cases, individual amino acids of the CDRscan be in close proximity to atoms of the target antigen. In someembodiments the CDR may be located in an immunoglobulin that may becomprised of three CDR regions. In some cases, as where there is morethan one CDR sequence in a larger amino acid sequence, the CDRs may beseparated by other sequences, and the CDRs numbered. In some cases,multiple CDRs are identified as CDR1, CDR2 and CDR3. Each CDR maycomprise amino acid residues from a Complementarity Determining Regionas defined by Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). Amino acid numbering ofCDRs, as well as other sequences within an antibody, or antibodyfragment is according to that of Kabat. In many cases, CDRs can bedefined by their position in a variable region sequence (numbering as inKabat), for example the light chain CDR 1 may comprise the amino acidsequence between position 24 and position 33; between position 50 andposition 56 for LC CDR2; and between position 89 and position 97 for LCCDR 3; and the heavy chain CDRs may lie between position 26 and position33 for CDR1; position 50 and position 66 for HC CDR 2; and betweenposition 97 and position 103 for HC CDR 3, and/or hypervariable loopsmay lie between light chain residues 26-32 (LC CDR1), residues 50-52 (LCCDR2) and residues 91-96 (LC CDR3); and heavy chain residues 26-32 (HCCDR1), residues 53-55 (HC CDR2) and residues 97-101 (HC CDR3). In someinstances, a Complementarity Determining Region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. In some embodiments, as in where the antibody is a single chainimmunoglobulin, there may be more than one CDR, more than two CDRs, morethan three CDRs, more than four CDRs, or more than five CDRs. In someembodiments, an antibody may be comprised of six CDRs.

“Framework regions,” FRs, are variable domain residues other than theCDR residues. In most embodiments a variable domain has between two andfour FRs identified sequentially. For example a variable regioncomprising three CDRs, has four FRs: FR1, FR2, FR3 and FR4. Where theCDRs are defined according to Kabat, the light chain FR residues arepositioned at about residues 1-23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3),and 98-107 (LCFR4) and the heavy chain FR residues are positioned aboutat residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96 (HCFR3), and 104-113(HCFR4) in the heavy chain residues. If the CDRs comprise amino acidresidues from hypervariable loops, the light chain FR residues arepositioned about at residues 1-23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3),and 98-107 (LCFR4) in the light chain and the heavy chain FR residuesare positioned about at residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96(HCFR3), and 104-113 (HCFR4) in the heavy chain residues. In someinstances, when the CDR comprises amino acids from both a CDR as definedby Kabat and those of a hypervariable loop, the FR residues will beadjusted accordingly. For example, when HC CDR1 includes amino acidsH26-H35, the heavy chain FR1 residues are at positions 1-25 and the FR2residues are at positions 36-49.

“Variable domain” refers to portions of a light chain and a heavy chainof traditional antibody molecule that includes amino acid sequences ofComplementarity Determining Regions (CDRs), and Framework Regions (FRs).VH refers to the variable domain of the heavy chain. VL refers to thevariable domain of the light chain.

“Fv” or “Fv fragment” refers to an antibody fragment which contains acomplete antigen recognition and binding site, comprising the FR and CDRsequences. In many embodiments, the Fv consists of a dimer of one heavyand one light chain variable domain in tight association, which can becovalent in nature, for example in a single chain Fv molecule (scFv).The three CDRs of each variable domain interact to define an antigenbinding site on the surface of the VH-VL polypeptide. Collectively, thesix CDRs or a subset thereof confer antigen binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has, in some cases,the ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

“Fab” or “Fab fragment” contains a variable and constant domain (CL) ofthe light chain and a variable domain and the first constant domain(CH1) of the heavy chain. F(ab′)₂ antibody fragments comprise a pair ofFab fragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. Sequence identity is then calculated relativeto the longer sequence, i.e. even if a shorter sequence shows 100%sequence identity with a portion of a longer sequence, the overallsequence identity will be less than 100%.

“Percent (%) amino acid sequence homology” is defined as the percentageof amino acid residues in a candidate sequence that are homologous withthe amino acid residues in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence homology. This method takes into account conservativesubstitutions. Conservative substitutions are those substitutions thatallow an amino acid to be substituted with a similar amino acid. Aminoacids can be similar in several characteristics, for example, size,shape, hydrophobicity, hydrophilicity, charge, isoelectric point,polarity, aromaticity, etc. Alignment for purposes of determiningpercent amino acid sequence homology can be achieved in various waysthat are within the ordinary skill of those persons of skill in the art.In some cases, amino acid sequences can be aligned using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. Sequence homology is then calculated relative to the longersequence, i.e. even if a shorter sequence shows 100% sequence identitywith a portion of a longer sequence, the overall sequence identity willbe less than 100%.

“Percent (%) nucleic acid sequence identity” is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in a reference sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Alignment for purposes of determining percentnucleic acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.Sequence identity is then calculated relative to the longer sequence,i.e. even if a shorter sequence shows 100% sequence identity with aportion of a longer sequence, the overall sequence identity will be lessthan 100%.

“Activity” or “biological activity” of a molecule can depend upon thetype of molecule and the availability of tests for assaying a givenactivity. For example, in the context of a Factor Bb antibody, activityrefers to its ability to partially or fully inhibit a biologicalactivity of Factor Bb, for example, binding to other complementproteins, serine protease activity, or MAC formation. A preferredbiological activity of the claimed Factor Bb antibody is the ability toachieve a measurable improvement in the state, e.g. pathology, of afactor Bb-associated disease or condition, such as, for example, acomplement-associated eye condition. In some cases, the activityinhibited by the disclosed anti-Factor Bb antibody is Factor Bb proteaseor cleavage activity. In other cases the activity is the ability to bindother complement proteins in a complex. In some embodiments, theactivity of the disclosed anti-Factor Bb antibody is measured by itsability to inhibit hemolysis. The activity can be determined through theuse of in vitro or in vivo tests, including binding assays, using arelevant animal model, or human clinical trials.

“Complement-associated eye condition” is used in the broadest sense andincludes all eye conditions the pathology of which involves complement,activated by either the classical, lectin, alternative or extrinsicpathways. Complement-associated eye conditions include, withoutlimitation, macular degenerative diseases, such as all stages ofage-related macular degeneration (AMD), including dry and exudative(non-exudative and exudative) forms, choroidal neovascularization (CNV),uveitis, diabetic and other ischemia-related retinopathies includingdiabetic macular edema, Central Retinal Vein Occlusion (CRVO), BranchedRetinal Vein Occlusion (BRVO), and other intraocular neovasculardiseases, such as diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, cornealneovascularization, and retinal neovascularization. A preferred group ofcomplement-associated eye conditions includes age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)AMD, choroidal neovascularization (CNV), Macular Telangiectasia,uveitis, diabetic and other ischemia-related neovascular-relatedretinopathies, or cellular degenerative diabetic macular edema,pathological myopia, von Hippel-Lindau disease, histoplasmosis of theeye, Doyne honeycomb retinal dystrophy/Malattia Leventinese, Stargartsdisease, Glucoma, Central Retinal Vein Occlusion (CRVO), BRVO, cornealneovascularization, retinal neovascularization.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopoeia for usein animals, and more particularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatpossesses the desired pharmacological activity of the parent compound.Such salts include acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; andsalts formed when an acidic proton present in the parent compound isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N methylglucamine, andthe like. In certain embodiments, a pharmaceutically acceptable salt isthe hydrochloride salt. In certain embodiments, a pharmaceuticallyacceptable salt is the sodium salt.

“Pharmaceutically acceptable excipient” refers to a pharmaceuticallyacceptable diluent, a pharmaceutically acceptable adjuvant, apharmaceutically acceptable vehicle, a pharmaceutically acceptablecarrier, or a combination of any of the foregoing with which a compoundprovided by the present disclosure can be administered to a patient,which does not destroy the pharmacological activity thereof and which isnon-toxic when administered in doses sufficient to provide atherapeutically effective amount of the compound or a pharmacologicallyactive metabolite thereof.

“Treatment” is an administration of at least one therapeutic agent forpreventing the development or altering the pathology of a disorder, oralleviating or lessening a symptom of a disorder. Accordingly, treatmentrefers to both therapeutic treatment and prophylactic or preventativemeasures. Those in need of treatment include those already with thedisorder as well as those in which the disorder is to be prevented. Asdisclosed herein, the preferred agent for administration comprises atleast one of the disclosed anti-Factor Bb antibodies. In treatment of acomplement related disease, the therapeutic agent, comprising at leastone of the presently disclosed antibodies or a coding sequence for suchantibody, may directly or indirectly alter the magnitude of response ofa component of the complement pathway, or render the disease moresusceptible to treatment by other therapeutic agents, e.g., antibiotics,antifungals, anti-inflammatory agents, chemotherapeutics, etc.

“Therapeutically effective amount” refers to the amount of an agentthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to effect suchtreatment of the disease or symptom thereof. The specifictherapeutically effective amount may vary depending, for example, on theagent, the disease and/or symptoms of the disease, severity of thedisease and/or symptoms of the disease, the age, weight, and/or healthof the patient to be treated, and the judgment of the prescribingphysician. An appropriate amount in any given compound can beascertained by those skilled in the art and/or is capable ofdetermination by routine experimentation.

“Therapeutically effective dose” refers to a dose that provideseffective treatment of a disease in a patient. A therapeuticallyeffective dose may vary from agent to agent and/or from patient topatient, and may depend upon factors such as the condition of thepatient and the severity of the disease. A therapeutically effectivedose can be determined in accordance with routine pharmacologicalprocedures known to those skilled in the art.

“Pathology” of a disease, such as a complement-associated eye condition,includes all phenomena that compromise the well-being of the patient.This includes, without limitation, abnormal or uncontrollable cellgrowth, protein production, abnormal or uncontrolled cell death,auto-antibody production, complement production, complement activation,MAC formation, interference with the normal functioning of neighboringcells, release of cytokines or other secretory products at abnormallevels, suppression or aggravation of any inflammatory or immunologicalresponse, infiltration of inflammatory cells into cellular spaces, etc.

“Mammal” as used herein refers to any animal classified as a mammal,including, without limitation, humans, higher primates, domestic andfarm animals, and zoo, sports or pet animals such horses, pigs, cattle,dogs, cats and ferrets, etc. In a preferred embodiment of the invention,the mammal is a human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

The present disclosure provides antibodies that bind Factor Bb protein.

The antibodies described herein comprise a scaffold structure with oneor more Complementarity Determining Regions (CDRs). In certainembodiments, the CDRs include no more than two amino acid additions,deletions, or substitutions from one or more of the heavy chain CDR1,CDR2, and CDR3, and the light chain CDR1, CDR2 and CDR3 of a parentsequence. In other embodiments, the CDRs are defined by a consensussequence having common conserved amino acid sequences and variable aminoacid sequences as described herein.

In certain embodiments, the scaffold structure of the Factor Bbantibodies of the disclosure can be based on antibodies, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (e.g. antibodymimetics), chimeric antibodies, humanized antibodies, antibody fusions(e.g. antibody conjugates), and fragments of each, respectively. Thevarious structures are further described and defined hereinbelow. TheFactor Bb antibodies are useful in treating consequences, symptoms,and/or the pathology associated with Factor Bb activity. These include,but are not limited to, atherosclerosis, ischemia-reperfusion followingacute myocardial infarction, Henoch-Schonlein purpura nephritis, immunecomplex vasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke,cardiomyopathy, hemorrhagic shock, crush injury, multiple organ failure,hypovolemic shock and intestinal ischemia, transplant rejection, cardiacSurgery, PTCA, spontaneous abortion, neuronal injury, spinal cordinjury, myasthenia gravis, Huntington's disease, amyotrophic lateralsclerosis, multiple sclerosis, Guillain Bane syndrome, Parkinson'sdisease, Alzheimer's disease, acute respiratory distress syndrome,asthma, chronic obstructive pulmonary disease, transfusion-related acutelung injury, acute lung injury, Goodpasture's disease, myocardialinfarction, post-cardiopulmonary bypass inflammation, cardiopulmonarybypass, septic shock, transplant rejection, xeno transplantation, burninjury, systemic lupus erythematosus, membranous nephritis, Berger'sdisease, psoriasis, pemphigoid, dermatomyositis, anti-phospholipidsyndrome, inflammatory bowel disease, hemodialysis, leukopheresis,plasmapheresis, heparin-induced extracorporeal membrane oxygenation LDLprecipitation, extracorporeal membrane oxygenation leukopheresis,plasmapheresis, heparin-induced extracorporeal membrane oxygenation LDLprecipitation, extracorporeal membrane oxygenation and the like.

Other uses for the disclosed antibodies include, for example, diagnosisof complement- and Factor Bb-associated diseases.

Aspects of the present disclosure provide Factor Bb antibodies,particularly antibodies that include at least one CDR including heavyand/or light CDRs, as more fully described below, or combinationsthereof.

In one aspect, the Factor Bb antibodies inhibit activity of Factor Bb,or inhibit the ability of Factor Bb to form protein complexes. Withoutbeing held to a particular mechanism or theory, in some embodiments theantibodies interrupt the complement pathway, thereby interrupting thecomplement cascade, formation of the MAC, and cell lysis. Thisdisruption may include, but is not limited to dry and wet (non-exudativeand exudative) AMD, choroidal neovascularization (CNV), uveitis,diabetic and other ischemia-related retinopathies, diabetic macularedema, pathological myopia, von Hippel-Lindau disease, histoplasmosis ofthe eye, Central Retinal Vein Occlusion (CRVO), cornealneovascularization, retinal neovascularization, and the like.

The antibodies of the disclosure thus may serve to identify conditionsrelated to the complement system or Factor Bb related diseases orconditions. In addition, the antibodies can be used to regulate and/orsuppress effects mediated by Factor B and/or other, downstream,complement proteins, as such having efficacy in the treatment andprevention of various diseases or conditions associated with complementand/or Factor Bb. This disruption may include, but is not limited toatherosclerosis, ischemia-reperfusion following acute myocardialinfarction, Henoch-Schonlein purpura nephritis, immune complexvasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke,cardiomyopathy, hemorrhagic shock, crush injury, multiple organ failure,hypovolemic shock and intestinal ischemia, transplant rejection, cardiacSurgery, PTCA, spontaneous abortion, neuronal injury, spinal cordinjury, myasthenia gravis, Huntington's disease, amyotrophic lateralsclerosis, multiple sclerosis, Guillain Bane syndrome, Parkinson'sdisease, Alzheimer's disease, acute respiratory distress syndrome,asthma, chronic obstructive pulmonary disease, transfusion-related acutelung injury, acute lung injury, Goodpasture's disease, myocardialinfarction, post-cardiopulmonary bypass inflammation, cardiopulmonarybypass, septic shock, transplant rejection, xeno transplantation, burninjury, systemic lupus erythematosus, membranous nephritis, Berger'sdisease, psoriasis, pemphigoid, dermatomyositis, anti-phospholipidsyndrome, inflammatory bowel disease, hemodialysis, leukopheresis,plasmapheresis, heparin-induced extracorporeal membrane oxygenation LDLprecipitation, extracorporeal membrane oxygenation leukopheresis,plasmapheresis, heparin-induced extracorporeal membrane oxygenation LDLprecipitation, extracorporeal membrane oxygenation, and the like.

More specifically, the disclosure provides anti-Factor Bb antibodies andpolynucleotides that encode them. In various aspects, the anti-Factor Bbantibodies inhibit at least one of the biological responses mediated bythe Factor Bb and/or other complement proteins, and as such can beuseful for ameliorating the effects of complement-associated and FactorBb-associated diseases or disorders. Also provided by the disclosure areexpression systems, including mammalian cell lines and bacterial cells,for the production of Factor Bb antibodies and methods of treatingdiseases associated with Factor Bb.

The antibodies of the present disclosure comprise a scaffold structureand one or more complementary determining regions (CDRs) that bind toFactor Bb. In one embodiment, an amino acid sequence comprises any ofSEQ ID NOs:1-6 or SEQ ID NOs: 18-23.

In various embodiments, the antibody comprises a first and/or secondamino acid sequence. In an embodiment, the first and/or the second aminoacid sequence is selected from the group consisting of SEQ ID NOs:8-15or SEQ ID NOs: 24-31.

In various embodiments, the antibodies can include one or both of thefirst and second amino acid sequences. The first and second amino acidsequences can be a single linear amino acid sequence, can be covalentlybonded by disulfide bridges, or can be non-covalently bonded.

Factor Bb

Complement factor B is a glycosylated protein composed of a single93,000 Da polypeptide chain encoded by the CFB gene. It is an essentialcomponent of the alternative pathway of complement activation and isfound in human plasma at approximately 200 μg/mL. In the presence of Mg′factor B binds to C3b and the C3b:B complex can be activated by factorD, a serine protease that circulates as an active trypsin-like serineprotease. Cleavage of factor B by factor D causes the release of the Bafragment (33,000 Da) and leaves the (60,000 Da) Bb fragment bound toC3b. This Bb subunit is a serine protease, called a C3 and a C5convertase because it converts both of these proteins to their activeforms by cleaving off the small peptides C3a and C5a, respectively.

Factor B is a tightly regulated, highly specific serine protease. In itsactivated form, it catalyzes the central amplification step ofcomplement activation to initiate inflammatory responses, cell lysis,phagocytosis and B-cell stimulation. Factor B is activated throughassembly with either surface-bound C3b, or its soluble counterpartC3(H₂O). After binding to C3, Factor B is cleaved by factor D into asmall fragment, Factor Ba (residues 1-234) and a large fragment, FactorBb (residues 235-739). Factor Ba dissociates from the complex, leavingbehind the alternative pathway C3 convertase complex C3b-Bb, whichcleaves C3 into C3a and C3b. The C3b-Bb protease complex is not stable,and once dissociated from the complex Factor Bb does not re-associatewith C3b.

The proenzyme factor B consists of three N-terminal complement controlprotein (CCP) domains, connected by a 45-residue linker to a VWA domainand a C-terminal serine protease (SP) domain, which carries thecatalytic center. Striking differences from other serine proteases areobserved in the active center of factor B. Factor Bb comprises theC-terminal serine protease domain, and the CCP domains are found inFactor Ba.

The amino acid sequence of the human Factor B is shown in SEQ ID NO:16.Other forms of Factor B that are useful in the present disclosureinclude mutants and variations that are at least 70% or at least 90%homologous to the human native Factor B sequence of SEQ ID NO:16.

The amino acid sequence of human Factor Bb is SEQ ID NO:7. Other formsof Factor Bb that are useful in the present disclosure include mutantsand variations that are at least 70% or at least 90% homologous to thenative Factor Bb sequence of SEQ ID NO:7.

The amino acid sequence of human Factor Ba is SEQ ID NO:17. Other formsof Factor Ba that are useful in the present disclosure include mutantsand variations that are at least 70% or at least 90% homologous to thenative Factor Ba sequence of SEQ ID NO:17.

Inhibiting Factor B, Factor Bb, or Factor Ba function/activity asdescribed herein represents inhibition of Factor Bb. One example ofassaying the complement alternative pathway is the Hemolysis Assay:Activation of the alternative pathway of (AP) requires higherconcentrations of serum than the classical pathway. Generally, a finalconcentration of 5 mM Mg⁺⁺ in the presence of 5 mM EGTA is used in theassays where the EGTA chelates Ca⁺⁺ preferentially. The AP of mostmammalian species is activated spontaneously by rabbit erythrocytes sothey are a convenient target. Prepare rabbit erythrocytes (ComplementTechnology, Inc.) by washing 3 times with GVB0 (CompTech product) andresuspending into 5×10⁸/ml. Different amount of anti-factor Bb antibodywas diluted with GVB0. Mix the 100 ul reaction on ice in the order ofserial diluted anti-factor Bb antibody, 0.1M MgEGTA (CompTech product),1/2NHS (normal human serum diluted 1/2 with GVB0), and rabbit Er. Then,incubate the reaction at 37° C. for 30 minutes on a shaker. Add 1.0 mlcold GVBE. Mix and centrifuge for 3 min at approx. 1000×g, or higher, topellet cells. Transfer 100 ul of the supernatant to a 96-well plate andread at 412 nm (SoftMax Pro 4.7.1). Data was analyzed using GraphPadPrism 6.

Factor Bb Antibodies.

In one aspect, the disclosure provides antibodies that bind Factor Bbwith greater affinity than they bind Factor B.

In certain aspects, the disclosure provides recombinant antibodies thatbind Factor Bb, i.e. Factor Bb antibodies or anti-Factor Bb antibodies.In this context, recombinant antibodies can be produced usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid as described below. Methods and techniques for theproduction of recombinant proteins are well known in the art.

In some embodiments, the antibodies of the disclosure are isolated orpurified. An isolated or purified antibody can be unaccompanied by atleast some of the material with which it is normally associated in itsnatural state (contaminating material). In a preferred embodiment, thecontaminating material constitutes less than about 50%, more preferablyless than about 20%, and more preferably less than about 10% by weightof the total weight of a given sample. In some embodiments thecontaminant may be a protein or peptide.

A pure protein comprises at least about 50% by weight of the totalprotein, with at least about 80% being preferred, and at least about 90%being particularly preferred. In many embodiments, the purifiedanti-Factor Bb antibody is produced from in or from an organism otherthan the organism from which it is derived. In some embodiments, theanti-Factor Bb antibody can be made at a significantly higherconcentration than is normally seen, through the use of an induciblepromoter or high expression promoter, such that the antibody is made atincreased concentration levels.

In some embodiments, the isolated or purified antibody can be removedfrom components that can interfere with diagnostic and/or therapeuticuses for the antibody. In preferred embodiments, the antibody will bepurified to greater than 90% by weight of antibody as determined by theLowry method, and most preferably more than 99% by weight, to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a common amino acid sequencing technique(e.g. Edman degradation and mass spectrometry), or to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor silver stain. Isolated antibodies include antibodies in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

The disclosed antibody can bind specifically to Factor Bb and can beused to inhibit or modulate the biological activity of Factor Bb. Incertain embodiments, the disclosed antibodies are created byimmunization of an animal, in other cases antibodies can be produced byrecombinant DNA techniques. In additional embodiments, anti-Factor Bbantibodies can be produced by enzymatic or chemical cleavage ofnaturally occurring antibodies. In some embodiments, the antibody cancomprise a tetramer. In some of these embodiments, each tetramer istypically composed of two identical pairs of polypeptide chains, eachpair having one light chain (typically having a molecular weight ofabout 25 kDa) and one heavy chain (typically having a molecular weightof about 50-70 kDa). The amino-terminal portion of each chain includes avariable region of about 100 to 110 or more amino acids and can beresponsible for antigen recognition. The carboxy-terminal portion ofeach chain can define a constant region, which is primarily responsiblefor effector function. Human light chains are classified as kappa andlambda light chains. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. IgG has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4.

Some naturally occurring antibodies, for example antibodies found incamels and llamas, can be dimers consisting of two heavy chains andinclude no light chains. Muldermans et al., 2001, J. Biotechnol.74:277-302; Desmyter et al., 2001, J. Biol. Chem. 276:26285-26290.Crystallographic studies of camel antibodies have revealed that the CDR3regions of these antibodies form a surface that interacts with theantigen and thus is critical for antigen binding like in the moretypical tetrameric antibodies. The disclosure encompasses dimericantibodies consisting of two heavy chains, or fragments thereof that canbind to and/or inhibit the biological activity of Factor Bb.

The antibodies of the disclosure specifically bind to a Factor Bbprotein, preferably a human Factor Bb. An antibody can specifically bindto a target antigen, when the antibody has a higher binding affinity forthat target antigen than for any other antigen or protein. Thus, theantibodies described herein bind with higher affinity to Factor Bb, thanto any other protein. Typically, the binding affinity is measure bydetermining an equilibrium binding constant, for example a K_(d) (orKd), or K_(a) (or Ka). In some embodiments the disclosed antibody bindsto a target antigen with a Kd from about 10⁻⁷ M to about 10⁻¹² M, orfrom about 10⁻⁸ M to about 10⁻¹¹ M, or from about 10⁻⁹ M to about 10⁻¹⁰M. In most cases, the Kd of the disclosed antibody for the a non-targetantigen can be higher than the Kd for the target antigen, for examplewhere the Kd for the target is 10⁻¹⁰ M and the Kd for the non-target is10⁻⁸ M In some cases the Kd for the other antigen is greater than 1× thetarget antigen Kd, greater than 2× the target antigen Kd, greater than3× the target antigen Kd, greater than 4× the target antigen Kd, greaterthan 5× the target antigen Kd, greater than 6× the target antigen Kd,greater than 7× the target antigen Kd, greater than 8× the targetantigen Kd, greater than 9× the target antigen Kd, greater than 10× thetarget antigen Kd (for example where the Kd of the antibody is X⁻⁰⁹ Mfor the target antigen, the Kd of the antibody for another antigen canbe 10× greater, or X⁻⁰⁸ M), or greater than 100× (for example where theKd of the antibody is X⁻¹⁰ M for the target antigen, the Kd of theantibody for another antigen can be 10× greater, or X⁻⁰⁸ M). In somecases, the equilibrium binding constant can be expressed as anequilibrium association constant, K_(a) or Ka.

The equilibrium binding constant, can be determined using variousmethods. In some cases, an equilibrium binding constant for thedisclosed antibody is determined by measuring on (k₁) and off (k⁻¹)rates in a protein binding assay. One exemplary method of determiningthe equilibrium binding constant is by Bio-Layer Interferometry BLI is alabel-free technology capable of determining binding kinetics insolution. In one exemplary method, an antibody can be a human IgG, andthe antibody can be captured by an Anti-human IgG Fc capture (AHC)biosensor tips (FortéBio, Menlo Park, Calif., USA) according to themanufacturer's directions. Other types of protein binding assaysinclude: Co-immunoprecipitation; Bimolecular fluorescencecomplementation; Affinity electrophoresis; Pull-down assays; Labeltransfer; The yeast two-hybrid screen; Phage display; in vivocrosslinking of protein complexes using photo-reactive amino acidanalogs; Tandem affinity purification; Chemical cross-linking; Chemicalcross-linking followed by high mass MALDI mass spectrometry; SPINE(Strepprotein interaction experiment); Quantitative immunoprecipitationcombined with knock-down; Proximity ligation assay Bio-LayerInterferometry; Dual polarisation interferometry; Static lightscattering; Dynamic light scattering; Surface plasmon resonance;Fluorescence polarization/anisotropy; fluorescence correlationspectroscopy; Fluorescence resonance energy transfer; Protein activitydetermination by NMR multi-nuclear relaxation measurements, or 2D-FT NMRspectroscopy in solutions, combined with nonlinear regression analysisof NMR relaxation or 2D-FT spectroscopy data sets; Protein-proteindocking; Isothermal Titration Calorimetry; and, MicroscaleThermophoresis.

In embodiments where the antibody is used for therapeutic applications,one characteristic of a Factor Bb antibody is that it can modulateand/or inhibit one or more biological activities of, or mediated by,Factor Bb. In this case, an antibody can bind specifically to Factor Bb,can substantially modulate the activity of Factor Bb, and/or can inhibitthe binding of Factor Bb to other proteins (e.g. Factor C3). In somecases, the antibody may inhibit the serine protease activity of FactorBb by at least about 20%, 40%, 60%, 80%, 85%, or more.

In many embodiments, Factor Bb activity, and the antibody's ability toinhibit that activity, is measured by analyzing lysis of red blood cellsin the presence of 10% human serum. Activation of the alternativepathway of (AP) requires higher concentrations of serum than theclassical pathway. Generally, a final concentration of 5 mM Mg⁺⁺ in thepresence of 5 mM EGTA is used in the assays where the EGTA chelates Ca⁺⁺preferentially. The AP of most mammalian species is activatedspontaneously by rabbit erythrocytes so they are a convenient target.Prepare rabbit erythrocytes (Complement Technology, Inc.) by washing 3times with GVB0 (CompTech product) and re-suspending into 5×10⁸/ml.Different amount of anti-factor Bb antibody was diluted with GVB0. Mixthe 100 ul reaction on ice in the order of serial diluted anti-factor Bbantibody, 0.1M MgEGTA (CompTech product), 1/2NHS (normal human serumdiluted 1/2 with GVB0), and rabbit Er. Then, incubate the reaction at37° C. for 30 minutes on a shaker. Add 1.0 ml cold GVBE. Mix andcentrifuge for 3 min at approx. 1000×g, or higher, to pellet cells.Transfer 100 ul of the supernatant to a 96-well plate and read at 412 nm(SoftMax Pro 4.7.1). Data was analysized using GraphPad Prism 6.

Not every antibody that specifically binds to an antigen can blockantigen binding to its normal ligand and thus inhibit or modulate thebiological effects of the antigen. As is known in the art, such aneffect can depend on what portion of the antigen the antibody binds to,and on both the absolute and the relative concentrations of the antigenand the antibody, in this case, a Factor Bb antibody. To be consideredcapable of inhibiting or modulating the biological activity of FactorBb, as meant herein, an antibody can be able, for example, to inhibitthe serine protease activity of Factor Bb or human serum mediatedhemolysis by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99%, ormore.

The concentration of an antibody required to inhibit Factor Bb activitycan vary widely and may depend upon how tightly the antibody binds toFactor Bb. For example, one molecule or less of an antibody per moleculeof Factor Bb can be sufficient to inhibit biological activity. In someembodiments, a ratio of Factor Bb antibody of about 1,000:1 to about1:1,000, including about 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20, 1:40,1:60, 1:100, 1:500, 1:1,000 or more can be required to inhibit thebiological activity of Factor Bb. In many cases, the ability to inhibitFactor Bb activity may depend upon the concentration of Factor Bb and/orthe concentration of Factor Bb antibody.

In some embodiments, the antibodies of the disclosure comprise (a) ascaffold, and (b) one or a plurality of CDRs, regions that aredeterminative to antigen binding specificity and affinity. ComplementaryDetermining Regions or CDRs, are regions of an antibody that constitutesthe major surface contact points for antigen binding. One or more CDRsare embedded in the scaffold structure of the antibody. The scaffoldstructure of the antibodies of the disclosure can be the framework of anantibody, or fragment or variant thereof, or can be completely syntheticin nature. The various scaffold structures of the antibodies of thedisclosure are further described herein.

In a preferred embodiment of the presently disclosed antibodies, theantibody can be a variant antibody having an amino acid sequence with atleast 75% amino acid sequence identity or similarity with the amino acidsequence of a parent antibody. For example, in some embodiments theheavy or light chain variable domain sequence of the variant antibody is75% identical to the heavy or light chain variable domain sequence ofthe parent antibody, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. In most cases, the variant antibody will have few or no changes inthe CDR sequence, and therefore, in most cases, will bind the targetantigen with a similar affinity. Identity or similarity with respect tothis sequence is defined herein as the percentage of amino acid residuesin the variant sequence that are identical (i.e. same residue) orsimilar (i.e. amino acid residue from the same group based on commonside-chain properties, see below) with the parent antibody amino acidsequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

CDRs

The antibodies of the disclosure include scaffold regions and one ormore CDRs. An antibody of the disclosure may have between one and sixCDRs (as typically do naturally occurring antibodies), for example, oneheavy chain CDR1 (“HC CDR1” or “HC CDR1”), and/or one heavy chain CDR2(“HC CDR2” or “HC CDR2”), and/or one heavy chain CDR3 (“HC CDR3” or “HCCDR3”), and/or one light chain CDR1 (“LC CDR1” or “LC CDR1”), and/or onelight chain CDR2 (“LC CDR2” or “LC CDR2”), and/or one light chain CDR3(“LC CDR3” or “LC CDR3”). The term “naturally occurring” as usedthroughout the specification in connection with biological materialssuch as polypeptides, nucleic acids, host cells, and the like, refers tomaterials which are found in nature. In naturally occurring antibodies,a heavy chain CDR1 typically comprises about five (5) to about seven (7)amino acids, a heavy chain CDR2 typically comprises about sixteen (16)to about nineteen (19) amino acids, and a heavy chain CDR3 typicallycomprises about three (3) to about twenty five (25) amino acids. CDR1 ofthe light chain typically comprises about ten (10) to about seventeen(17) amino acids, the light chain CDR2 typically comprises about seven(7) amino acids, and the light chain CDR3 typically comprises aboutseven (7) to about ten (10) amino acids.

Amino acids of the present disclosure include natural and syntheticamino acids (e.g., homophenylalanine, citrulline, ornithine, andnorleucine). Such synthetic amino acids can be incorporated, inparticular when the antibody is synthesized in vitro by conventionalmethods well known in the art. In addition, any combination ofpeptidomimetic, synthetic and naturally occurring residues/structurescan be used. Amino acid includes imino acid residues such as proline andhydroxyproline. The amino acid “R group” or “side chain” can be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration. In some embodiments,the amino acids can form peptidomimetic structures, i.e., peptide orprotein analogs, such as peptoids (see, Simon et al., 1992, Proc. Natl.Acad. Sci. U.S.A. 89:9367, incorporated by reference herein), which canbe resistant to proteases or other physiological and/or storageconditions.

The structure and properties of CDRs within a naturally occurringantibody are described further below. Briefly, in a traditional antibodyscaffold, the CDRs are embedded within a framework in the heavy andlight chain variable region where they constitute the regionsresponsible for antigen binding and recognition. A variable regioncomprises at least three heavy or light chain CDRs, (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Public Health ServiceN.I.H., Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342: 877-883), within aframework region (designated framework regions 1-4, FR1, FR2, FR3, andFR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987). The CDRsprovided by the present disclosure, however, may not only be used todefine the antigen binding domain of a traditional antibody structure,but can be embedded in a variety of other scaffold structures, asdescribed herein.

Alanine Scanning was used to identify amino acid positions in the CDRsequences that, when modified, alter the binding affinity of anti-FactorBb antibodies.

Specific CDRs for use in the disclosed antibodies are presented in Table1, underlined amino acids are those where substitution to alaninesubstantially decreased binding.

TABLE 1 HC_(A) CDR1 GDIFSSHW SEQ ID NO: 1 HC_(A) CDR2 EILPRSGITHYNENFNGSEQ ID NO: 2 HC_(A) CDR3 AINWEDS SEQ ID NO: 3 LC_(A) CDR1 HASQNVNVWL SEQID NO: 4 LC_(A) CDR2 KASNLHT SEQ ID NO: 5 LC_(A) CDR3 QQGQSYPYT SEQ IDNO: 6 HC_(B) CDR1 DYYMS SEQ ID NO: 18 HC_(B) CDR2 FSRHRVYGYTPEYSASVKGSEQ ID NO: 19 HC_(B) CDR3 DNPGYYAMDY SEQ ID NO: 20 LC_(B) CDR1KASQSVDYDGDSYMN SEQ ID NO: 21 LC_(B) CDR2 AASNLES SEQ ID NO: 22 LC_(B)CDR3 QQSNADPYT SEQ ID NO: 23

The sequences for Factor B, Factor Ba and Factor Bb are shown in Table2.

TABLE 2 Factor Bb (SEQ ID NO: 7)KIVLDPSGSMNIYLVLDGSDSIGASNFTGAKKCLVNLIEKVASYGVKPRYGLVTYATYPKIWVKVSEADSSNADWVTKQLNEINYEDHKLKSGTNTKKALQAVYSMMSWPDDVPPEGWNRTRHVIILMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPREDYLDVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDVFYQMIDESQSLSLCGMVWEHRKGTDYHKQPWQAKISVIRPSKGHESCMGAVVSEYFVLTAAHCFTVDDKEHSIKVSVGGEKRDLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLKYGQTIRPICLPCTEGTTRALRLPPTTTCQQQKEELLPAQDIKALFVSEEEKKLTRKEVYIKNGDKKGSCERDAQYAPGYDKVKDISEVVTPRFLCTGGVSPYADPNTCRGDSGGPLIVHKRSRFIQVGVISWGVVDVCKNQKRQKQVPAHARDFHINLFQVLPWLKEKLQDE DLGFL Factor B (SEQID NO: 16) signal peptide underlined MGSNLSPQLCLMPFILGLLSGGVTTTPWSLARPQGSCSLEGVEIKGGSFRLLQEGQALEYVCPSGFYPYPVQTRTCRSTGSWSTLKTQDQKTVRKAECRAIHCPRPHDFENGEYWPRSPYYNVSDEISFHCYDGYTLRGSANRTCQVNGRWSGQTAICDNGAGYCSNPGIPIGTRKVGSQYRLEDSVTYHCSRGLTLRGSQRRTCQEGGSWSGTEPSCQDSFMYDTPQEVAEAFLSSLTETIEGVDAEDGHGPGEQQKRKIVLDPSGSMNIYLVLDGSDSIGASNFTGAKKCLVNLIEKVASYGVKPRYGLVTYATYPKIWVKVSEADSSNADWVTKQLNEINYEDHKLKSGTNTKKALQAVYSMMSWPDDVPPEGWNRTRHVIILMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPREDYLDVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDVFYQMIDESQSLSLCGMVWEHRKGLDYHKQPWQAKISVIRPSKGHESCMGAVVSEYFVLTAAHCFTVDDKEHSIKVSVGGEKRDLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLKYGQTIRPICLPCTEGTTRALRLPPTTTCQQQKEELLPAQDIKALFVSEEEKKLTRKEVYIKNGDKKGSCERDAQYAPGYDKVKDISEVVTPRFLCTGGVSPYADPNTCRGDSGGPLIVHKRSRFIQVGVISWGVVDVCKNQKRQKQVPAHARDFHINLFQVLPWLKEKLQDEDLGFL Factor Ba (SEQ ID NO: 17) Signalpeptide underlined MGSNLSPQLCLMPFILGLLSGGVTTTPWSLARPQGSCSLEGVEIKGGSFRLLQEGQALEYVCPSGFYPYPVQTRTCRSTGSWSTLKTQDQKTVRKAECRAIHCPRPHDFENGEYWPRSPYYNVSDEISFHCYDGYTLRGSANRTCQVNGRWSGQTAICDNGAGYCSNPGIPIGTRKVGSQYRLEDSVTYHCSRGLTLRGSQRRTCQEGGSWSGTEPSCQDSFMYDTPQEVAEAFLSSLTETIEGVDAEDG HGPGEQQKR

In another embodiment, the disclosure provides an antibody that bindsFactor Bb (SEQ ID NO:7), wherein said antibody comprises at least one HCCDR region having no more than two (2) amino acid additions, deletionsor substitutions of any of SEQ ID NOs:1-3 or SEQ ID NOs:18-20, and/or atleast one the LC CDR region having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:4-6 or SEQ IDNOs:21-23. The various heavy chain and light chain variable regions ofthe disclosure are depicted in TABLE 3 and SEQ ID NOs:8-15 or SEQ IDNOs:24-31. In some embodiments, of particular use are antibodies with aHC CDR3 or LC CDR3 region. Additionally, in some embodiments antibodiescan have one CDR having no more than two (2) amino acid additions,deletions or substitutions of the sequence selected from the HC CDRregions of any of SEQ ID NOs:1-3 or SEQ ID NOs:18-20 and a LC CDR havingno more than two (2) amino acid additions, deletions, or substitutionsof any of SEQ ID NOs:4-6 or SEQ ID NOs:21-23 (e.g., the antibody has twoCDR regions, one HC CDR and one LC CDR, a specific embodiment areantibodies with both a HC CDR3 and a LC CDR3, for example, SEQ ID NOs:3and 6).

TABLE 3 Light Chain Sequences L1_(A) (SEQ ID NO: 8)DIQMTQSPSSLSASVGDRVTITCRASQNVNVWLSWYQQKPGKAPKLLIFKASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L2_(A)(SEQ ID NO: 9) DIQMTQSPSSLSASVGDTVTITCRASQNVNVWLSWYQQKPGKAPKLLIFKASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L3_(A)(SEQ ID NO: 10) DIQMTQSPSSLSASVGDTVTITCRASQNVNVWLSWYQQKPGKAPKLLIFKAGNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L4_(A)(SEQ ID NO: 11) DIQMTQSPSSLSASVGDTVTITCRASQSVNVWLSWYQQKPGKAPKLLIFKAGNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC L1_(B)(SEQ ID NO: 24) EIVLTQSPATLSLSPGERATLSCKASQSVDYDGDSYMNWYQQKPGQAPRLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNADPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECL2_(B) (SEQ ID NO: 25)EIVLTQSPATLSLSPGERATLSCKASQSVDYDGDSYMNWYQQKPGQAPRLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDGATYYCQQSNADPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECL3_(B) (SEQ ID NO: 26)EIVLTQSPATLSLSPGERATLGCKASQSVDYDGDSYMNWYQQKPGQAPRLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNADPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECL4_(B) (SEQ ID NO: 27 EIVLTQSPATLSLSPGERATLGCKASQSVDYDGDSYMNWYQQKPGQAPRLLIYAASNRESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNADPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHeavy Chain Sequences H1_(A) (SEQ ID NO: 12)QVQLVQSGAEVKKPGSSVKVSCKASGDIFSSHWIEWIRQAPGQGLEWMGEILPRSGITNYAQKFQGRVTFTADTSTSTAYMELSSLRSEDTAVYYCAINWEDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H2_(A) (SEQ ID NO: 13)QVQLVQSGAEVKKPGSSVKVSCKASGDIFSSHWIEWIRQAPGQGLEWMGEILPRSGITHYAEKFQGRVTFTADTSTSTAYMELSSLRSEDTAVYYCAINWEDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H3_(A) (SEQ ID NO: 14)QVQLVQSGAEVKKPGSSVKVSCKADGDIFSSHWIEWIRQAPGQGLEWMGEILPRSGITHYAEKFQGRVTFTADTSTSTAYMELSSLRSEDTAVYYCAINWEDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H4_(A) (SEQ ID NO: 15)QVQLVQSGAEVKKPGSSVKVSCKADGDIFSSHWIEWVRQAPGQGLEWMGEILPRSGITNYAEKFQGRVTFTADTSTSTAYMELSSLRSEDTAVYYCAINWEDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H1_(B) (SEQ ID NO: 28)EVQLVESGGGLVQPGGSLRLSCATSGFTFRDYYMSWVRQAPGKGLEWVGFSRHRVYGYTTEYAASVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCARDNPGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H2_(B) (SEQ ID NO: 29)EVQLVESGGGLVQPGGSLRLSCATSGFTFRDYYMSWVRQAPGKGLEWLGFSRHRVYGYTPEYAASVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCARDNPGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H3_(B) (SEQ ID NO: 30)EVQLVESGGGLVQPGGSLRLSCGTTGFTFRDYYMSWVRQAPGKGLEWLGFSRHRVYGYTPEYAASVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCARDNPGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL H4_(B) (SEQ ID NO: 31)EVQLVESGGGLVQPGGSLRLSCGTTGFTFRDYYMSWVRQAPGKGLEWLGFSRHRAYGYTPEYAASVKGRFTISRDNSKNTLYLQMNSLKTEDTAVYYCARDNPGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

Variant CDR Sequences

An additional aspect of the disclosure provides for an isolated antibodythat binds Factor Bb, wherein the isolated antibody comprises a heavychain amino acid sequence having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:12-15 or SEQID NOs:28-31, or a light chain amino acid sequence having no more thantwo (2) amino acid additions, deletions or substitutions of any of SEQID NOs:8-11 or SEQ ID NOs:24-27.

A further aspect of the disclosure provides for an isolated antibodythat binds Factor Bb, wherein the isolated antibody comprises a heavychain amino acid sequence having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:12-15 or SEQID NOs:28-31, and a light chain amino acid sequence having no more thantwo (2) amino acid additions, deletions or substitutions of any of SEQID NOs:8-11 or SEQ ID NOs:24-27. It is noted that any of the heavy chainsequences can be mixed and matched with any of the light chainsequences.

In another embodiment, the disclosure provides an antibody that binds aFactor Bb, wherein said antibody comprises at least one HC CDR regionhaving no more than two (2) amino acid additions, deletions orsubstitutions of any HC CDR1, HC CDR2, or HC CDR3 region (as discussedabove) of SEQ ID NOs:1-3 or SEQ ID NOs:18-20 and/or at least one LC CDRregion having no more than two (2) amino acid additions, deletions orsubstitutions of any LC CDR1, LC CDR2, or LC CDR3 region (as discussedabove) of SEQ ID NOs:4-6 or SEQ ID NOs:21-23. In this embodiment, ofparticular use are antibodies with a HC CDR3 or LC CDR3 region.Additional embodiments utilize antibodies with one CDR having no morethan 2 amino acid additions, deletions or substitutions of the sequenceselected from the HC CDR regions of any of SEQ ID NOs:1-3 or SEQ IDNOs:18-20 and a LC CDR region having no more than two (2) amino acidadditions, deletions or substitutions of any of SEQ ID NOs:4-6 or SEQ IDNOs:21-23 (e.g., the antibody has two CDR regions, one HC CDR and one LCCDR, a specific embodiment are antibodies with both a HC CDR3 and a LCCDR3 region, for example SEQ ID NO:3 and 6).

As will be appreciated by those in the art, for any antibody with morethan one CDR from the depicted sequences, any combination of CDRsindependently selected from the depicted sequences is useful. Thus,antibodies with one, two, three, four, five or six of independentlyselected CDRs can be generated. However, as will be appreciated by thosein the art, specific embodiments generally utilize combinations of CDRsthat are non-repetitive, e.g., antibodies are generally not made withtwo HC CDR2 regions, etc.

An additional aspect of the disclosure provides for an isolated antibodythat binds Factor Bb where the isolated antibody comprises a heavy chainamino acid sequence having no more than two (2) amino acid additions,deletions or substitutions of any of SEQ ID NOs:12-15 or SEQ IDNOs:28-31, or a light chain amino acid sequence having no more than two(2) amino acid additions, deletions or substitutions of any of SEQ IDNOs:8-11 or SEQ ID NOs:24-27.

A further aspect of the disclosure provides for an isolated antibodythat binds Factor Bb where the isolated antibody comprises a heavy chainamino acid sequence having no more than two (2) amino acid additions,deletions or substitutions of any of SEQ ID NOs:12-15 or SEQ IDNOs:28-31, and a light chain amino acid sequence having no more than two(2) amino acid additions, deletions or substitutions of any of SEQ IDNOs:8-11 or SEQ ID NOs:24-27. It is noted that any of the heavy chainsequences can be mixed and matched with any of the light chainsequences.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs, described herein, is at least 80% when comparedto the sequences disclosed herein. In many cases the aa homology,similarity, or identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, and 99%.

Sequence Identity/Homology

As it is known in the art, a number of different programs can be used toidentify the degree of sequence identity or similarity a protein ornucleic acid has to a known sequence.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values for proteins:overlap span=1, overlap fraction=0.125, word threshold, T=11. The HSP Sand HSP S2 parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values can be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenindividual variant CDRs or variable regions are at least 80% to thesequences depicted herein, and more typically with preferably increasinghomologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and almost 100%.

In a similar manner, percent (%) nucleic acid sequence identity, withrespect to the nucleic acid sequence of the disclosed antibodies, is thepercentage of nucleotide residues in a candidate sequence that areidentical with the nucleotide residues in the coding sequence of theantibody. A specific method utilizes the BLASTN module of WU-BLAST-2 setto the default parameters, with overlap span and overlap fraction set to1 and 0.125, respectively.

Generally, the nucleic acid sequence homology, similarity, or identitybetween the nucleotide sequences encoding individual variant CDRs andvariant variable domain sequences are at least 80%, and more typicallywith preferably increasing homologies or identities of at least 85%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%.In many cases non-identical nucleic acid sequences, because of thedegeneracy of the genetic code, can code for the same amino acidsequence.

Homology between nucleotide sequences is often defined by their abilityto hybridize to each other. In some embodiments, selective hybridizationcan refer to binding with high specificity. Polynucleotides,oligonucleotides and fragments thereof in accordance with the disclosureselectively hybridize to nucleic acid strands under hybridization andwash conditions that minimize appreciable amounts of detectable bindingto nonspecific nucleic acids. High stringency conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein.

The stringency of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA tore-anneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

High stringency conditions are known in the art; see, for exampleSambrook et al., 2001, supra, and Short Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, both ofwhich are hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques In Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993).

In some embodiments, stringent or high stringency conditions can beidentified by those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42 C; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextransulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium (as the target sequences are present in excess,at Tm, 50% of the probes are occupied at equilibrium). Stringentconditions will be those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium Ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short probes (e.g., 10 to 50 nucleotides) andat least about 60° C. for long probes (e.g., greater than 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions can be used, as areknown in the art; see, Sambrook et al., 2001, supra; Ausubel et al.,1992, supra, and Tijssen, 1993, supra.

In some cases, moderately stringent conditions can include the use ofwashing solution and hybridization conditions (e.g., temperature, ionicstrength and % SDS) less stringent that those described above. Anexample of moderately stringent conditions is overnight incubation at37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as probe lengthand the like.

In some embodiments, the disclosed antibodies and variants thereof canbe prepared by site specific mutagenesis of nucleotides within a DNAsequence encoding the antibody. This can be achieved using cassette orPCR mutagenesis or other techniques well known in the art, to produceDNA encoding the variant, and thereafter expressing the recombinant DNAin cell culture as outlined herein. In some cases, antibody fragmentscomprising variant CDRs having up to about 100-150 residues can beprepared by in vitro synthesis using established techniques. Thesevariant fragments can exhibit the same qualitative biological activityas the naturally occurring analogue, e.g., binding to Factor Bb andinhibiting complement, although variants can also be selected which havemodified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the a mutation at agiven site, random mutagenesis can be conducted at the target codon orregion and the expressed antibody CDR or variable region sequencevariants screened for the optimal desired antibody activity. Techniquesfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known, for example, M13 primer mutagenesis andPCR mutagenesis. Screening of the mutants is done using assays ofantibody activities, such as Factor Bb binding.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions can betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions can be much larger.

Substitutions, deletions, insertions or any combination thereof can beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of theantibody. However, larger changes can be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as Table 4.

TABLE 4 Original Exemplary Residue Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Set Trp Tyr Tyr Trp, Phe Val Ile, Leu

Changes in function or immunological identity can be made by selectingsubstitutions that are less conservative than those shown in Table 4.For example, substitutions can be made which more significantly affect:the structure of the polypeptide backbone in the area of the alteration,for example the alpha-helical or beta-sheet structure; the charge orhydrophobicity of the molecule at the target site; or the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the polypeptide's properties are those in which(a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the disclosed Factor Bb antibody, as needed.Alternatively, variant can be selected wherein the biological activityof the disclosed antibody is altered. For example, glycosylation sitescan be altered or removed as discussed herein.

Disclosed herein are polypeptide sequences homologous to SEQ ID NOs:1-6and 8-15 and SEQ ID NOs:18-23 and SEQ ID NOs:24-31. Polypeptidesdisclosed herein can include amino acid sequences that are identical tothe disclosed amino acid sequences. In other cases, the claimedpolypeptides include amino acid sequences that can comprise conservativeamino acid substitutions as compared to the disclosed sequence.Conservative amino acid substitutions can include amino acids that sharecharacteristics with the substituted amino acid. In various cases,conservative substitution can be made without significant change in thestructure or function of the polypeptide.

Conservative amino acid substitutions can be made on the basis ofrelative similarity of side-chain, size, charge, hydrophobicity,hydrophilicity, isoelectric point, etc. In various cases, substitutionscan be assayed for their effect on the function of the protein byroutine testing. Conserved amino acid substitutions include amino acidswith similar hydrophilicity value, as wherein amino acids have ahydropathic index which can be based upon an amino acid's hydrophobicityand charge. In various cases, conserved amino acid substitutions can bemade between amino acids of the same class, for example non-polar aminoacids, acidic amino acids, basic amino acids, and neutral amino acids.Conservative substitutions can also be based upon size or volume. Aminoacids can also be classified based upon their ability to form or break agiven structure, such as an alpha helix, beta sheet, or intra- orinter-molecular interaction. In various cases conservative amino acidsubstitutions are based upon more than one characteristic.

Currently disclosed polypeptides can include both natural andnon-natural amino acids. In various cases, natural amino acid sidechains can be substituted with non-natural side chains. In variouscases, amino acids can be derivatized.

The disclosed polypeptides include polypeptides that are homologous tothe sequences of SEQ ID NO:1-6 and 8-15 and SEQ ID NO:18-31. Homologycan be expressed as % identity or % similarity or % positive. In variouscases, % identity is a percentage of amino acids that are identicalbetween two aligned polypeptides, and % similar or % positive is apercentage of amino acids that are non-identical but representconservative substitutions. A conservative substitution may be asubstitution of a like-charged amino acid, a like-sized amino acid, alike-polarity amino acid, etc. For example, lysine to arginine can beconsidered a conservative substitution where charge is considered.

In various cases, two polypeptides can be aligned by algorithms, forexample BLASTp. In various cases, the BLASTp parameters can be set witha maximum target sequence length equal to, greater, or less than thelength of the longer of the two polypeptides, the expect threshold canbe set to 10, the word size to 3, and scoring matrix can be BLOSUM62,with gap costs of 11 for existence and 1 for extension. BLASTp canreport homology of aligned polypeptides as “Identities” and “Positives.”The aligned sequences can include gaps to achieve the alignment.

In various cases, homology of amino acid sequences can reflect thepercentage of identity or positives when optimally aligned as describedabove. In various cases, the % homology (% positive) or % identity canbe calculated by dividing the number of aligned amino acids within acomparison window. A comparison window can be the entire length of oneor the other polypeptides, if the two polypeptides are of unequallength. In other cases, the comparison window can be a portion of one ofthe polypeptides. In various cases the comparison window for measuringhomology or identity of two polypeptide sequences is greater than about40 aa (amino acids), 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70 aa, 75 aa, 80aa, 85 aa, 90 aa, 95 aa, 100 aa, 150 aa, or 200 aa, and/or less thanabout 200 aa, 150 aa, 100 aa, 95 aa, 90 aa, 85 aa, 80 aa, 75 aa, 70 aa,65 aa, 60 aa, 55 aa, 50 aa, or 45 aa. In some embodiment, as in the casewith CDR sequences, the comparison window may be less than 40 aa, forexample between less than about 25 aa, 24 aa, 23 aa, 22 aa, 21 aa, 20aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10aa, 9 aa, 8 aa, 7 aa, 6 aa, 5 aa, or 4 aa, and greater than about 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, or 24 aa.

In various cases, the claimed amino acid sequences can have % identityor % homology (% positive) over a given comparison window, that isgreater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% and/or less than about 100%, 99%, 98%,97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70%.

In various cases, a sequence alignment can be performed using variousalgorithms, including dynamic, local, and global alignment. For example,the algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482; thealignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443;the similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad.Sci. USA 85: 2444. In various cases, computer programs can implementthese algorithms (such as EMBOSS, GAP, BESTFIT, FASTA, TFASTA BLAST,BLOSUM, etc.).

In alternative cases, conserved amino acid substitutions can be madewhere an amino acid residue is substituted for another in the sameclass, for example where the amino acids are divided into non-polar,acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu,Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His;neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

In some cases, conserved amino acid substitutions can be made where anamino acid residue is substituted for another having a similarhydrophilicity value (e.g., within a value of plus or minus 2.0), wherethe following can be an amino acid having a hydropathic index of about−1.6 such as Tyr (−1.3) or Pro (−1.6)s are assigned to amino acidresidues: Arg (+3;0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3);Asn (+0.2); Gin (+0.2); Gly (0); Pro (−0.5); Thr (−0.4); Ala (−0.5); His(−0.5); Cys (−1.0); Met (−1.3); Val (−1.5); Leu (−1.8); Ile (−1.8); Tyr(−2.3); Phe (−2.5); and Trp (−3.4).

In alternative cases, conserved amino acid substitutions can be madewhere an amino acid residue is substituted for another having a similarhydropathic index (e.g., within a value of plus or minus 2.0). In suchcases, each amino acid residue can be assigned a hydropathic index onthe basis of its hydrophobicity and charge characteristics, as follows:lie (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9);Ala (+1.8); Gly (−0.4); Thr (−0.7); Ser (−0.8); Trp (−0.9); Tyr (−1.3);Pro (−1.6); His (−3.2); Glu (−3.5); Gln (−3.5); Asp (−3.5); Asn (−3.5);Lys (−3.9); and Arg (−4.5).

In alternative cases, conservative amino acid changes include changesbased on considerations of hydrophilicity or hydrophobicity, size orvolume, or charge. Amino acids can be generally characterized ashydrophobic or hydrophilic, depending primarily on the properties of theamino acid side chain. A hydrophobic amino acid exhibits ahydrophobicity of greater than zero, and a hydrophilic amino acidexhibits a hydrophilicity of less than zero, based on the normalizedconsensus hydrophobicity scale of Eisenberg et al. (J. Mol. Bio.179:125-142, 184). Genetically encoded hydrophobic amino acids includeGly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically encodedhydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, andLys. Non-genetically encoded hydrophobic amino acids includet-butylalanine, while non-genetically encoded hydrophilic amino acidsinclude citrulline and homocysteine.

Hydrophobic or hydrophilic amino acids can be further subdivided basedon the characteristics of their side chains. For example, an aromaticamino acid is a hydrophobic amino acid with a side chain containing atleast one aromatic or heteroaromatic ring, which can contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR,etc., where R is independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl,(C₀-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl,substituted (C₀-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₀-C₂₀) aryl,(C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe, Tyr, and Trp.

An non-polar or apolar amino acid is a hydrophobic amino acid with aside chain that is uncharged at physiological pH and which has bonds inwhich a pair of electrons shared in common by two atoms is generallyheld equally by each of the two atoms (i.e., the side chain is notpolar). Genetically encoded apolar amino acids include Gly, Leu, Val,Ile, Ala, and Met. Apolar amino acids can be further subdivided toinclude aliphatic amino acids, which is a hydrophobic amino acid havingan aliphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala, Leu, Val, and Ile.

A polar amino acid is a hydrophilic amino acid with a side chain that isuncharged at physiological pH, but which has one bond in which the pairof electrons shared in common by two atoms is held more closely by oneof the atoms. Genetically encoded polar amino acids include Ser, Thr,Asn, and Gln.

An acidic amino acid is a hydrophilic amino acid with a side chain pKavalue of less than 7. Acidic amino acids typically have negativelycharged side chains at physiological pH due to loss of a hydrogen ion.Genetically encoded acidic amino acids include Asp and Glu. A basicamino acid is a hydrophilic amino acid with a side chain pKa value ofgreater than 7. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include Arg, Lys, and His.

A % amino acid sequence identity value is determined by the number ofmatching identical residues divided by the total number of residues ofthe “longer” sequence in the comparison window. The “longer” sequence isthe one having the most actual residues in the comparison window (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment can include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the protein encoded by the sequence the disclosedpolypeptide, it is understood that in one case, the percentage ofsequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. In percentidentity calculations relative weight is not assigned to variousmanifestations of sequence variation, such as, insertions, deletions,substitutions, etc.

In one case, only identities are scored positively (+1) and all forms ofsequence variation including gaps are assigned a value of “0”, whichobviates the need for a weighted scale or parameters as described belowfor sequence similarity calculations. Percent sequence identity can becalculated, for example, by dividing the number of matching identicalresidues by the total number of residues of the “shorter” sequence inthe aligned region and multiplying by 100. The “longer” sequence is theone having the most actual residues in the aligned region.

Scaffolds

As noted herein, the antibodies of the present disclosure can comprise ascaffold structure into which the CDR(s) described above can be grafted.In one embodiment, the scaffold structure is a traditional antibodystructure, that is, an antibody comprising two heavy and two light chainvariable domain sequences. In some cases, the antibody combinationsdescribed herein can include additional components (framework, J and Dregions, constant regions, etc.) that make up a heavy and/or a lightchain. Some embodiments include the use of human scaffold components.

Accordingly, in various embodiments, the antibodies of the disclosurecomprise the scaffolds of traditional antibodies. In some embodiments,the disclosed antibodies can be human and monoclonal antibodies,bispecific antibodies, diabodies, minibodies, domain antibodies,synthetic antibodies, chimeric antibodies, antibody fusions, andfragments of each, respectively. The above described CDRs andcombinations of CDRs can be grafted into any of the following scaffolds.

Chimeric antibodies of the present disclosure can comprise a heavyand/or light chain sequence that is identical or homologous to thecorresponding sequences derived from a particular species. For example,in one embodiment the anti-Factor Bb antibody is a chimeric antibodycomprising a human Fc domain, while the remainder of the antibody can beidentical or homologous to corresponding mouse or rodent sequences.Chimeric antibodies can be fragments of such antibodies, so long as thefragments exhibit the desired biological activity and comprise sequencethat is derived from another species, class of antibody, or subclass ofantibody (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc.Natl. Acad. Sci. USA 81:6851-6855).

In some embodiments, a variable region of the presently disclosedanti-Factor Bb antibody comprises at least three heavy or light chainCDRs, see, supra (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, Public Health Service N.I.H., Bethesda, Md.; seealso Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al.,1989, Nature 342: 877-883), embedded within a framework region(designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat etal., 1991, supra; see also Chothia and Lesk, 1987, supra).

In some cases, the antibody can be comprised of a heavy chain variabledomain sequence or a light chain variable domain sequence. In some casesthe heavy or light chain variable domain sequence may comprise asequence selected from the sequences of Table 3.

Traditional antibody structural units, in most cases, comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one light chain (typically having amolecular weight of about 25 kDa) and one heavy chain (typically havinga molecular weight of about 50-70 kDa). The amino-terminal portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal portion of each chain defines a constant region, whilethe heavy chain may comprise a total of three constant regions (CH1,CH2, and CH3), wherein the constant regions may aid in regulatingeffector function. Human light chains are classified as kappa and lambdalight chains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. IgG has several subclasses, including, but notlimited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including,but not limited to, IgM1 and IgM2.

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about twelve (12) or more amino acids, withthe heavy chain also including a “D” region of about ten (10) more aminoacids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch.7, 2nd ed. Raven Press, N.Y. The variable regions of each light andheavy chain pair form the antibody binding site.

Some naturally occurring antibodies, for example found in camels andllamas, are dimers consisting of two heavy chains and include no lightchains. Muldermans et al., 2001, J. Biotechnol. 74:277-302; Desmyter etal., 2001, J. Biol. Chem. 276:26285-26290. Crystallographic studies of acamel antibody have revealed that the CDR3 regions form a surface thatinteracts with the antigen and thus is critical for antigen binding likein the more typical tetrameric antibodies. The disclosure encompassesdimeric antibodies consisting of two heavy chains, or fragments thereof,that can bind to and/or inhibit the biological activity of Factor Bb.

The variable regions of the heavy and light chains typically exhibit thesame general structure of relatively conserved framework regions (FR)joined by three complementarity determining regions or CDRs. The CDRscomprise hypervariable regions of an antibody that are responsible forantigen recognition and binding. The CDRs from the two chains of eachpair are aligned and supported by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest. Chothia et al., 1987, J. Mol. Biol. 196:901-917; Chothia etal., 1989, Nature 342:878-883.

CDRs constitute the major surface contact points for antigen binding.See, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. Further,CDR3 of the light chain and, especially, CDR3 of the heavy chain mayconstitute the most important determinants in antigen binding within thelight and heavy chain variable regions. See, e.g., Chothia and Lesk,1987, supra; Desiderio et al., 2001, J. Mol. Biol. 310:603-615; Xu andDavis, 2000, Immunity 13:37-45; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290; and Muyldermans, 2001, J. Biotechnol. 74:277-302. Insome antibodies, the heavy chain CDR3 appears to constitute the majorarea of contact between the antigen and the antibody. Desmyter et al.,2001, supra. In vitro selection schemes in which CDR3 alone is variedcan be used to vary the binding properties of an antibody. Muyldermans,2001, supra; Desiderio et al., 2001, supra.

Naturally occurring antibodies typically include a signal sequence,which directs the antibody into the cellular pathway for proteinsecretion and which is not present in the mature antibody. Apolynucleotide encoding an antibody of the disclosure may encode anaturally occurring signal sequence or a heterologous signal sequence asdescribed below.

In one embodiment, the anti-Factor Bb antibody is a monoclonal antibody,with from one (1) to six (6) of the CDRs, as outlined herein. Theantibodies of the disclosure can be of any type including IgM, IgG(including IgG1, IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. Inspecific embodiment, the antibody is an IgG type antibody. In an evenmore specific embodiment, the antibody is an IgG2 type antibody.

In some embodiments, the antibody can comprise complete heavy and lightchains, the CDRs are all from the same species, e.g., human.Alternatively, for example in embodiments wherein the antibody containsless than six CDRs from the sequences outlined above, additional CDRscan be either from other species (e.g., murine CDRs), or can bedifferent human CDRs than those depicted in the sequences. For example,human HC CDR3 and LC CDR3 regions from the appropriate sequencesidentified herein can be used, with HC CDR1, HC CDR2, LC CDR1 and LCCDR2 being optionally selected from alternate species, or differenthuman antibody sequences, or combinations thereof. For example, the CDRsof the disclosure can replace the CDR regions of commercially relevantchimeric or humanized antibodies.

Specific embodiments utilize scaffold components of the antibodies thatare human components.

In some embodiments, however, the scaffold components can be a mixturefrom different species. As such, the antibody can be a chimeric antibodyand/or a humanized antibody. In general, both chimeric antibodies andhumanized antibodies can be antibodies that combine regions or aminoacids from more than one species. For example, chimeric antibodies, inmost embodiments, comprise variable region(s) from a mouse, rat, rabbit,or other suitable non-human animal, and the constant region(s) from ahuman.

Humanized antibodies are antibodies that are originally derived fromnon-human antibodies, for example a mouse antibody. In variousembodiments of a humanized anti-Factor Bb antibody, the variable-domainframework regions or framework amino acids, which are derived from anon-human antibody, can be changed to be amino acid identities found atcorresponding positions in human antibodies. In some embodiments of ahumanized antibody, the entire antibody, except the CDRs, can be encodedby a polynucleotide of human origin or is identical to such an antibodyexcept within its CDRs. In other embodiments, a humanized antibody maycomprise specific amino acid positions whose identity has been changedto the identity of the same or similar position in a corresponding humanantibody. The CDRs, some or all of which can be encoded by nucleic acidsoriginating in a non-human organism, are grafted into the beta-sheetframework of a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones, 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. In some embodiments, the CDRs can be human, and thus bothhumanized and chimeric antibodies in this context include some non-humanCDRs. In some cases, humanized antibodies can be generated that comprisethe HC CDR3 and LC CDR3 regions, with one or more of the other CDRregions being of a different special origin.

In one embodiment, the Factor Bb antibody can be a multispecificantibody, and notably a bispecific antibody, (e.g. diabodies). These areantibodies that bind to two (or more) different antigens, for exampleFactor Bb, and another antigen, or two different epitopes of Factor Bb.Diabodies can be manufactured in a variety of ways known in the art(Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449),e.g., prepared chemically or from hybrid hybridomas.

In one embodiment, the Factor Bb antibody is a minibody. Minibodies areminimized antibody-like proteins comprising a scFv joined to a CH3domain. Hu et al., 1996, Cancer Res. 56:3055-3061.

In one embodiment, the Factor Bb antibody is a domain antibody; see, forexample U.S. Pat. No. 6,248,516. Domain antibodies (dAbs) are functionalbinding domains of antibodies, corresponding to the variable regions ofeither the heavy (VH) or light (VL) chains of human antibodies dABs havea molecular weight of approximately 13 kDa, or less than one-tenth thesize of a full antibody. dABs are well expressed in a variety of hostsincluding bacterial, yeast, and mammalian cell systems. In addition,dAbs are highly stable and retain activity even after being subjected toharsh conditions, such as freeze-drying or heat denaturation. See, forexample, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; USSerial No. 2004/0110941; European Patent 0368684; U.S. Pat. No.6,696,245, WO04/058821, WO04/003019 and WO03/002609, all incorporatedentirely by reference.

In one embodiment, the Factor Bb antibody is an antibody fragment, thatis a fragment of any of the antibodies outlined herein that retainbinding specificity to Factor Bb. In various embodiments, the antibodiesare a F(ab), F(ab′), F(ab′)2, Fv, or a single chain Fv fragments. At aminimum, an antibody, as meant herein, comprises a polypeptide that canbind specifically to an antigen, wherein the polypeptide comprises allor part of a light and/or a heavy chain variable region.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546) which consists of a singlevariable, (v) isolated CDR regions, (vi) F(ab′)₂ fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) diabodies ortriabodies, multivalent or multispecific fragments constructed by genefusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448). The antibody fragments can be modified. For example, themolecules can be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245). Again, as outlined herein, the non-CDR components ofthese fragments are preferably human sequences.

In one embodiment, the Factor Bb antibody is a traditional antibody, forexample a human immunoglobulin. In this embodiment, as outlined above,specific structures comprise complete heavy and light chains depictedcomprising the CDR regions. Additional embodiments utilize one or moreof the CDRs of the disclosure, with the other CDRs, framework regions, Jand D regions, constant regions, etc., coming from other humanantibodies. For example, the CDRs of the disclosure can replace the CDRsof any number of human antibodies, particularly commercially relevantantibodies.

In one embodiment, the Factor Bb antibody is an antibody fusion protein(e.g. an antibody conjugate). In this embodiment, the antibody is fusedto a conjugation partner. The conjugate partner can be proteinaceous ornon-proteinaceous; the latter generally being generated using functionalgroups on the antibody (see the discussion on covalent modifications ofthe antibodies) and on the conjugate partner. For example linkers areknown in the art; for example, homo- or hetero-bifunctional linkers asare well known (see, Pierce Chemical Company catalog, technical sectionon cross-linkers, pages 155-200, incorporated herein by reference).

In one embodiment, the Factor Bb antibody is an antibody analog. In somecases antibody analogs can be referred to as synthetic antibodies. Forexample, a variety of recent work utilizes either alternative proteinscaffolds or artificial scaffolds with grafted CDRs. Such scaffoldsinclude, but are not limited to, mutations introduced to stabilize thethree-dimensional structure of the antibody as well as wholly syntheticscaffolds consisting for example of biocompatible polymers. See, forexample, Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(PAMs) can be used, as well as work based on antibody mimetics utilizingfibronectin components as a scaffold.

VH and VL Variants

As outlined above, in some embodiments the disclosure providesantibodies comprising, or consisting of a heavy chain variable regioncomprising SEQ ID NO:1-3 and/or a light chain variable region of SEQ IDNO:4-6, respectively, or fragments thereof as defined above. Thus, inthose embodiments, the antibody comprises not only at least one CDR orvariant, but also at least part of a depicted framework sequence. Inaddition, the disclosure encompasses variants of such heavy chainvariable sequences or light chain variable sequences.

A variant variable region, generally shares an amino acid homology,similarity, or identity of at least 80% with those a parent variableregion, such as those disclosed herein. In some embodiments, the variantand parent sequence homologies or identities are at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and almost 100%. Thenucleic acid sequence homology, similarity, or identity between thenucleotide sequences encoding individual variant VHs and VLs and thenucleic acid sequences depicted herein are at least 70% with thosedepicted herein, and more typically with preferably increasinghomologies or identities of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% and almost 100%. In addition, a variant variableregion can, in many embodiments, shares the biological function,including, but not limited to, at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the specificity and/or activity of the parentCDR. In some case, homology and/or identity is only measured outside theCDR sequences, which can be identical. In other cases, the homologyand/or identity is measured throughout the entire sequence, includingCDR sequences. In some embodiments, constant region variants may also beincluded.

In various cases, homology of amino acid sequences can reflect thepercentage of identity or positives when optimally aligned as describedabove. In various cases, the % homology (% positive) or % identity canbe calculated by dividing the number of aligned amino acids within acomparison window. A comparison window can be the entire length of oneor the other polypeptides, if the two polypeptides are of unequallength. In other cases, the comparison window can be a portion of one ofthe polypeptides. In various cases the comparison window for measuringhomology or identity of two polypeptide sequences is greater than about40 aa (amino acids), 45 aa, 50 aa, 55 aa, 60 aa, 65 aa, 70 aa, 75 aa, 80aa, 85 aa, 90 aa, 95 aa, 100 aa, 150 aa, or 200 aa, and/or less thanabout 200 aa, 150 aa, 100 aa, 95 aa, 90 aa, 85 aa, 80 aa, 75 aa, 70 aa,65 aa, 60 aa, 55 aa, 50 aa, or 45 aa. In some embodiment, as in the casewith CDR sequences, the comparison window may be less than 40 aa, forexample between less than about 25 aa, 24 aa, 23 aa, 22 aa, 21 aa, 20aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10aa, 9 aa, 8 aa, 7 aa, 6 aa, 5 aa, or 4 aa, and greater than about 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, or 24 aa.

In various cases, the claimed amino acid sequences can have % identityor % homology (% positive) over a given comparison window, that isgreater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% and/or less than about 100%, 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, or 75%.

Covalent Modifications of Anti-Factor Bb Antibodies

Covalent modifications of antibodies are included within the scope ofthis disclosure, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues can be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to form0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisdisclosure.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Glycosylation

Another type of covalent modification of the antibodies included withinthe scope of this disclosure comprises altering the glycosylationpattern of the protein. As is known in the art, glycosylation patternscan depend on both the sequence of the protein (e.g., the presence orabsence of particular glycosylation amino acid residues, discussedbelow), or the host cell or organism in which the protein is produced.Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the disclosed antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tri-peptide sequences(for N-linked glycosylation sites). The alteration may also be made bythe addition of, or substitution by, one or more serine or threonineresidues to the starting sequence (for O-linked glycosylation sites).For ease, the antibody's amino acid sequence is preferably alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the target polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) can be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antibody can beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites can be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

PEGylation

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 and/or 4,179,337,all incorporated entirely by reference. In addition, as is known in theart, amino acid substitutions can be made in various positions withinthe antibody to facilitate the addition of polymers such as PEG.

Labels

In some embodiments, the covalent modification of the antibodies of thedisclosure comprises the addition of one or more labels.

The term “labelling group” means any detectable label. Examples ofsuitable labelling groups include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentgroups, biotinyl groups, or predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, the labelling group is coupled to theantibody via spacer arms of various lengths to reduce potential sterichindrance. Various methods for labelling proteins are known in the artand can be used in performing the present disclosure.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which can beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and can be used in performing the present disclosure.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

A fluorescent label can be any molecule that can be detected via itsinherent fluorescent properties. Suitable fluorescent labels include,but are not limited to, fluorescein, rhodamine, tetramethylrhodamine,eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS,BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, theAlexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, Rhodamine,and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham LifeScience, Pittsburgh, Pa.). Suitable optical dyes, includingfluorophores, are described in Molecular Probes Handbook by Richard P.Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558), all incorporated entirely by reference.

Polynucleotides Encoding Anti-Factor Bb Antibodies

In certain aspects, the disclosure provides nucleic acid moleculesencoding the antibodies described herein. In some cases the disclosednucleic acids code for IgGs, variable regions, or CDRs described herein.Nucleic acids include both DNA and RNA molecules. Nucleic acids can beeither natural or synthetic nucleic acids. Nucleic acids of the presentdisclosure are typically polynucleic acids; that is, polymers ofindividual nucleotides that are covalently joined by 3′, 5′phosphodiester bonds. In various cases the nucleotide sequences can besingle-stranded, double stranded, or a combination thereof. In somevariations, the nucleotide sequences can comprise natural nucleic acids,synthetic nucleic acids, non-natural nucleic acids, and/or nucleic acidanalogs. The nucleotide sequences can further comprise other non-nucleicacid molecules such as amino acids, and other monomers.

In many embodiments, the coding sequence may be an isolated nucleic acidmolecule. The isolated nucleic acid molecule is identified and separatedfrom at least one component with which it is ordinarily associated inthe natural source. In some cases a component can be a nucleotidesequence, protein, or non-proteinaceous molecule. An isolatedanti-Factor Bb antibody-encoding nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated anti-FactorBb antibody-encoding nucleic acid molecules therefore are distinguishedfrom the encoding nucleic acid molecule(s) as they exist in naturalcells. However, an isolated anti-Factor Bb antibody-encoding nucleicacid molecule includes anti-Factor Bb antibody-encoding nucleic acidmolecules contained in cells that ordinarily express anti-Factor Bbantibody where, for example, the nucleic acid molecule is in achromosomal location different from that of natural cells. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in an organism. However, in some cases an isolatednucleic acid molecule can be a nucleic acid contained within a cell, forexample, wherein the isolated nucleic acid molecule is introduced into acell and resides in either an extrachromosomal location or in achromosomal location different from its native location.

Depending on its use, the nucleic acid can be double stranded, singlestranded, or contain portions of both double stranded or single strandedsequence. As will be appreciated by those in the art, the depiction of asingle strand (“Watson”) also defines the sequence of the other strand(“Crick”). A recombinant nucleic can be a nucleic acid, originallyformed in vitro, in general, by the manipulation of nucleic acid byendonucleases, in a form not normally found in nature. Thus an isolatedantibody can be encoded by a nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis disclosure. It is understood that once a recombinant nucleic acid,with all necessary control elements, is made and reintroduced into ahost cell or organism, it can replicate non-recombinantly, i.e., usingthe in vivo cellular machinery of the host cell rather than in vitromanipulations; however, such nucleic acids, once produced recombinantly,although subsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the disclosure.

In some embodiments, the recombinant nucleic acid may comprise one ormore control elements or control sequences. Control element and controlsequence refers to nucleic acid sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers. As used herein, an operablylinked sequence, is a nucleic acid sequence in a functional relationshipwith another nucleic acid sequence. For example, nucleic acid codingsequences can be operably linked to nucleic acid control sequences. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation. Inmost embodiments, an operably linked sequence is a DNA sequencecovalently linked to, for example, a secretory leader sequence. In manyembodiments, enhancer sequences are not required to be adjacent to acoding sequence, rather the two sequences may be separated by one ormore nucleic acids.

In various cases, the nucleic acids of the disclosed nucleotidesequences can include nucleotides that are metabolized in a mannersimilar to naturally occurring nucleotides. Also included arenucleic-acid-like structures with synthetic backbone analoguesincluding, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs)(see, e.g.: “Oligonucleotides and Analogues, a Practical Approach,”edited by F. Eckstein, IRL Press at Oxford University Press (1991);“Antisense Strategies,” Annals of the New York Academy of Sciences,Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J.Med. Chem. 36:1923-1937; and “Antisense Research and Applications”(1993, CRC Press)). PNAs contain non-ionic backbones, such asN-(2-aminoethyl) glycine units. Phosphorothioate linkages are describedin: WO 97/03211; WO 96/39154; and Mata (1997) Toxicol. Appl. Pharmacol.144:189-197. Other synthetic backbones encompassed by this term includemethyl-phosphonate linkages or alternating methyl-phosphonate andphosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzyl-phosphonate linkages (Samstag (1996) AntisenseNucleic Acid Drug Dev 6: 153-156).

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids can be made,all of which encode the CDRs (and heavy and light chains or othercomponents of the antibody) of the present disclosure. Thus, havingidentified a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids, by simply modifyingthe sequence of one or more codons in a way which does not change theamino acid sequence of the encoded protein.

In various cases, nucleotide sequences encoding the polypeptidesequences of SEQ ID NO:1-6 and 8-15 and 18-31 are included. Thesenucleotide coding sequences can be translated into a polypeptide havingan amino acid sequence identical to the disclosed polypeptide sequence.In many cases, nucleotides coding for identical polypeptides, may nothave identical nucleotide sequences. This is due to the degeneracy ofthe genetic code. The disclosed coding sequences can further compriseuntranslated sequences, for example polyadenylation sequences. Theinventive coding sequences can also comprise intron or intervening,non-translated, sequence that are spliced out of a transcribed mRNAprior to translation. In various cases the transcribed mRNA can becapped with a terminal 7-methylguanosine. In some embodiments, thecoding sequences will include coding sequences for amino acids that donot appear in the final antibody, for example sequences required forexport of the antibody.

In some variations, due to the degeneracy of the genetic code, multiplenucleotide coding sequences can encode the same polypeptide sequence.These inventive nucleic acid coding sequences can also be homologous tonucleotide sequences that encode the disclosed polypeptides. Thenucleotide coding sequences can be aligned by BLASTn, as describedabove. In various cases the homology (or identities in BLASTn) of thesealigned nucleotide sequences can be greater than about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and/or less than about100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, or 45%. Invarious cases, the homologous aligned sequences can be less than about700 nt, 600 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 90 nt, 80 nt, 70nt, 60 nt, 50 nt or 40 nt, and/or more than about 50 nt, 60 nt, 70 nt,80 nt, 90 nt, 100 nt, 200 nt, 300 nt, 400 nt, 500 nt, or 600 nt.

In various cases, the coding sequence directs transcription of aribonucleic acid sequence that can be translated into amino acidsequence according to the standard genetic code. In various cases, thecode can include variations to the canonical code. In some variations,the coding sequence can include introns, or intervening sequences thatdo not code for amino acids, but can be transcribed and later removedbefore the ribonucleic acid is translated into a polypeptide.

Methods of Producing Antibodies

The present disclosure also provides expression systems and constructsin the form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the disclosure provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas flanking sequences in certain embodiments will typically include oneor more of the following nucleotide sequences: a promoter, one or moreenhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the Factor Bbantibody coding sequence; the oligonucleotide sequence can encode apolyHis tag (such as hexaHis), or another “tag” such as FLAG, HA(hemaglutinin influenza virus), or myc, for which commercially availableantibodies exist. This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification or detection of the Factor Bb antibody from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed from the purified FactorBb antibody by various means such as using certain peptidases forcleavage.

Flanking sequences can be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence can be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this disclosure can beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence can be known. Here, the flanking sequence can be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it canbe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence can be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationcan be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one can be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, glutaminesynthetase (GS) and the tetracycline resistance gene. Advantageously, aneomycin resistance gene may also be used for selection in bothprokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as anantibody that binds to a Factor Bb polypeptide or Factor Bb epitope. Asa result, increased quantities of a polypeptide such as a Factor Bbantibody are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the disclosure will typically containa promoter that is recognized by the host organism and operably linkedto the molecule encoding the Factor Bb antibody. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown. A suitable promoter is operably linked to the DNA encoding heavychain or light chain comprising a Factor Bb antibody of the disclosureby removing the promoter from the source DNA by restriction enzymedigestion and inserting the desired promoter sequence into the vector.

In some embodiment, yeast cells may be used to produce the presentlydisclosed Factor Bb antibodies. Suitable promoters for use with yeasthosts are also well known in the art. Yeast enhancers are advantageouslyused with yeast promoters. Suitable promoters for use with mammalianhost cells are well known and include, but are not limited to, thoseobtained from the genomes of viruses such as polyoma virus, fowlpoxvirus, adenovirus (such as Adenovirus 2), bovine papilloma virus, aviansarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40). Other suitable mammalian promotersinclude heterologous mammalian promoters, for example, heat-shockpromoters and the actin promoter.

Additional promoters which can be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence can be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising aFactor Bb antibody of the disclosure by higher eukaryotes. Enhancers arecis-acting elements of DNA, usually about 10-300 bp in length, that acton the promoter to increase transcription. Enhancers are relativelyorientation and position independent, having been found at positionsboth 5′ and 3′ to the transcription unit. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus is used. The SV40 enhancer, the cytomegalovirusearly promoter enhancer, the polyoma enhancer, and adenovirus enhancersknown in the art are exemplary enhancing elements for the activation ofeukaryotic promoters. While an enhancer can be positioned in the vectoreither 5′ or 3′ to a coding sequence, it is typically located at a site5′ from the promoter. A sequence encoding an appropriate native orheterologous signal sequence (leader sequence or signal peptide) can beincorporated into an expression vector, to promote extracellularsecretion of the antibody. The choice of signal peptide or leaderdepends on the type of host cells in which the antibody is to beproduced, and a heterologous signal sequence can replace the nativesignal sequence. Examples of signal peptides that are functional inmammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

Expression vectors, for expressing the presently claimed antibodies ofthe disclosure can be constructed from a starting vector such as acommercially available vector. Such vectors may or may not contain allof the desired flanking sequences. Where one or more of the flankingsequences described herein are not already present in the vector, theycan be individually obtained and ligated into the vector. Methods usedfor obtaining each of the flanking sequences are well known to oneskilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain, a heavy chain, or a light chain and a heavy chaincomprising an Factor Bb antigen binding sequence has been inserted intothe proper site of the vector, the completed vector can be inserted intoa suitable host cell for amplification and/or polypeptide expression.The transformation of an expression vector for a Factor Bb antibody intoa selected host cell can be accomplished by well known methods includingtransfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes aFactor Bb antibody that can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity (such as glycosylation or phosphorylation) andease of folding into a biologically active molecule. A host cell can beeukaryotic or prokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC), includingbut not limited to Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines can be selected throughdetermining which cell lines have high expression levels andconstitutively produce antibodies with Factor Bb binding properties. Inanother embodiment, a cell line from the B cell lineage that does notmake its own antibody but has a capacity to make and secrete aheterologous antibody can be selected.

Use of Factor Bb Antibodies for Diagnostic and Therapeutic Purposes

Antibodies of the disclosure are useful for detecting Factor Bb inbiological samples and identification of cells or tissues that produceFactor Bb protein. For example, the Factor Bb antibodies of thedisclosure can be used in diagnostic assays, e.g., binding assays todetect and/or quantify Factor Bb expressed in a tissue or cell.Increased levels of Factor Bb may be an indication of diseases such asocular disorders, cancer, infection, and/or ulcerative colitis.Decreased levels of Factor Bb may be in indication of cirrhosis,glomerulonephritis, hereditary angioedema, hepatitis, kidney transplantrejection, lupus nephritis, malnutrition, and/or systemic lupuserythematosis.

In some embodiments, the antibodies of the disclosure that specificallybind to Factor Bb can be used in treatment of Factor Bb mediateddiseases in a patient in need thereof. In addition, the Factor Bbantibody of the disclosure can be used to inhibit Factor Bb from forminga complex with other complement proteins, thereby modulating thebiological activity of Factor Bb in a cell or tissue. Antibodies thatbind to Factor Bb thus can modulate and/or block interaction with otherbinding compounds and as such may have therapeutic use in amelioratingFactor Bb mediated diseases.

In some embodiments, Factor Bb antibodies may block the proteaseactivity of Factor Bb. In some cases, the binding of Factor Bb by FactorBb antibodies may result in disruption of the Factor Bb induced signaltransduction cascade.

Diagnostic Methods

The antibodies of the disclosure can be used for diagnostic purposes todetect, diagnose, or monitor diseases and/or conditions associated withFactor Bb or Factor B. The disclosure provides for the detection of thepresence of Factor Bb in a sample using classical immunohistologicalmethods known to those of skill in the art (e.g., Tijssen, 1993,Practice and Theory of Enzyme Immunoassays, vol 15 (Eds R. H. Burdon andP. H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.);Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al.,1987, J. Cell Biol. 105:3087-3096). The detection of Factor Bb can beperformed in vivo or in vitro.

Diagnostic applications provided herein include use of the antibodies todetect expression of Factor Bb and/or binding to Factor Bb. Examples ofmethods useful in the detection of the presence of Factor Bb includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA).

For diagnostic applications, the antibody typically can be labeled witha detectable labeling group. Suitable labeling groups include, but arenot limited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent groups (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent groups, biotinyl groups, or predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and can be used in performing the present disclosure.

One aspect of the disclosure provides for identifying a cell or cellsthat express Factor Bb. In a specific embodiment, the antibody islabeled with a labeling group and the binding of the labeled antibody toFactor Bb is detected. In a further specific embodiment, the binding ofthe antibody to Factor Bb can be detected in vivo. In a further specificembodiment, the antibody/Factor Bb complex is isolated and measuredusing techniques known in the art. See, for example, Harlow and Lane,1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed.1991 and periodic supplements); John E. Coligan, ed., 1993, CurrentProtocols In Immunology New York: John Wiley & Sons.

Another aspect of the disclosure provides for detecting the presence ofa test molecule that competes for binding to Factor Bb with theantibodies of the disclosure. An example of one such assay would involvedetecting the amount of free antibody in a solution containing an amountof Factor Bb in the presence or absence of the test molecule. Anincrease in the amount of free antibody (i.e., the antibody not bound toFactor Bb) would indicate that the test molecule is capable of competingfor Factor Bb binding with the antibody. In one embodiment, the antibodyis labeled with a labeling group. Alternatively, the test molecule islabeled and the amount of free test molecule is monitored in thepresence and absence of an antibody.

Indications

The complement system has been implicated in contributing to severalacute and chronic conditions, including atherosclerosis,ischemia-reperfusion following acute myocardial infarction,Henoch-Schonlein purpura nephritis, immune complex vasculitis,rheumatoid arthritis, arteritis, aneurysm, stroke, cardiomyopathy,hemorrhagic shock, crush injury, multiple organ failure, hypovolemicshock and intestinal ischemia, transplant rejection, cardiac Surgery,PTCA, spontaneous abortion, neuronal injury, spinal cord injury,myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis,multiple sclerosis, Guillain Bane syndrome, Parkinson's disease,Alzheimer's disease, acute respiratory distress syndrome, asthma,chronic obstructive pulmonary disease, transfusion-related acute lunginjury, acute lung injury, Goodpasture's disease, myocardial infarction,post-cardiopulmonary bypass inflammation, cardiopulmonary bypass, septicshock, transplant rejection, xeno transplantation, burn injury, systemiclupus erythematosus, membranous nephritis, Berger's disease, psoriasis,pemphigoid, dermatomyositis, anti-phospholipid syndrome, inflammatorybowel disease, hemodialysis, leukopheresis, plasmapheresis,heparin-induced extracorporeal membrane oxygenation LDL precipitation,extracorporeal membrane oxygenation, and macular degeneration.

Macular degenerative diseases, such as all stages of age-related maculardegeneration (AMD), including dry and wet (non-exudative and exudative)forms, choroidal neovascularization (CNV), uveitis, diabetic and otherischemia-related retinopathies, and other intraocular neovasculardiseases, such as diabetic macular edema, pathological myopia, vonHippel-Lindau disease, histoplasmosis of the eye, Central Retinal VeinOcclusion (CRVO), Branched Retinal Vein Occlusion (BRVO), cornealneovascularization, and retinal neovascularization. A preferred group ofcomplement-associated eye conditions includes age-related maculardegeneration (AMD), including non-exudative (wet) and exudative (dry oratrophic) AMD, choroidal neovascularization (CNV), diabetic retinopathy(DR), and endophthalmitis.

The presently disclosed anti-Factor Bb antibodies can be used incombination with one or more cytokines, lymphokines, hematopoieticfactor(s), and/or an anti-inflammatory agent.

Treatment of the diseases and disorders recited herein can include theuse of first line drugs for control of pain and inflammation incombination (pretreatment, post-treatment, or concurrent treatment) withtreatment with one or more of the antibodies provided herein. Thesedrugs are classified as non-steroidal, anti-inflammatory drugs (NSAIDs).Secondary treatments include corticosteroids, slow acting antirheumaticdrugs (SAARDs), or disease modifying (DM) drugs. Information regardingthe following compounds can be found in The Merck Manual of Diagnosisand Therapy, Sixteenth Edition, Merck, Sharp & Dohme ResearchLaboratories, Merck & Co., Rahway, N.J. (1992) and in Pharmaprojects,PJB Publications Ltd.

In a specific embodiment, the present disclosure is directed to the useof an antibody and any of one or more NSAIDs for the treatment of thediseases and disorders recited herein. NSAIDs owe theiranti-inflammatory action, at least in part, to the inhibition ofprostaglandin synthesis (Goodman and Gilman in “The PharmacologicalBasis of Therapeutics,” MacMillan 7th Edition (1985)). NSAIDs can becharacterized into at least nine groups: (1) salicylic acid derivatives;(2) propionic acid derivatives; (3) acetic acid derivatives; (4) fenamicacid derivatives; (5) carboxylic acid derivatives; (6) butyric acidderivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more salicylic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. Such salicylic acid derivatives, prodrug esters andpharmaceutically acceptable salts thereof comprise: acetaminosalol,aloxiprin, aspirin, benorylate, bromosaligenin, calciumacetylsalicylate, choline magnesium trisalicylate, magnesium salicylate,choline salicylate, diflusinal, etersalate, fendosal, gentisic acid,glycol salicylate, imidazole salicylate, lysine acetylsalicylate,mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine,parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,salicylamide 0-acetic acid, salsalate, sodium salicylate andsulfasalazine. Structurally related salicylic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more propionic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The propionic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: alminoprofen,benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen,flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen,ibuprofen aluminum, ibuproxam, indoprofen, isoprofen, ketoprofen,loxoprofen, miroprofen, naproxen, naproxen sodium, oxaprozin,piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid,pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen. Structurallyrelated propionic acid derivatives having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In yet another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more acetic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The acetic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: acemetacin,alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin,diclofenac potassium, diclofenac sodium, etodolac, felbinac,fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac,glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac,metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin,sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium,zidometacin and zomepirac. Structurally related acetic acid derivativeshaving similar analgesic and anti-inflammatory properties are alsointended to be encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more fenamic acid derivatives,prodrug esters or pharmaceutically acceptable salts thereof. The fenamicacid derivatives, prodrug esters and pharmaceutically acceptable saltsthereof comprise: enfenamic acid, etofenamate, flufenamic acid,isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid,mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamicacid and ufenamate. Structurally related fenamic acid derivatives havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more carboxylic acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The carboxylic acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof which can be used comprise:clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine.Structurally related carboxylic acid derivatives having similaranalgesic and anti-inflammatory properties are also intended to beencompassed by this group.

In yet another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more butyric acidderivatives, prodrug esters or pharmaceutically acceptable saltsthereof. The butyric acid derivatives, prodrug esters, andpharmaceutically acceptable salts thereof comprise: bumadizon,butibufen, fenbufen and xenbucin. Structurally related butyric acidderivatives having similar analgesic and anti-inflammatory propertiesare also intended to be encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more oxicams, prodrug esters,or pharmaceutically acceptable salts thereof. The oxicams, prodrugesters, and pharmaceutically acceptable salts thereof comprise:droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide.Structurally related oxicams having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more pyrazoles, prodrugesters, or pharmaceutically acceptable salts thereof. The pyrazoles,prodrug esters, and pharmaceutically acceptable salts thereof which canbe used comprise: difenamizole and epirizole. Structurally relatedpyrazoles having similar analgesic and anti-inflammatory properties arealso intended to be encompassed by this group.

In an additional specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatmentor, concurrent treatment) with any of one or more pyrazolones, prodrugesters, or pharmaceutically acceptable salts thereof. The pyrazolones,prodrug esters and pharmaceutically acceptable salts thereof which canbe used comprise: apazone, azapropazone, benzpiperylon, feprazone,mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone,propylphenazone, ramifenazone, suxibuzone and thiazolinobutazone.Structurally related pyrazalones having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more of the following NSAIDs:ε-acetamidocaproic acid, S-adenosyl-methionine, 3-amino-4-hydroxybutyricacid, amixetrine, anitrazafen, antrafenine, bendazac, bendazac lysinate,benzydamine, beprozin, broperamole, bucolome, bufezolac, ciproquazone,cloximate, dazidamine, deboxamet, detomidine, difenpiramide,difenpyramide, difisalamine, ditazol, emorfazone, fanetizole mesylate,fenflumizole, floctafenine, flumizole, flunixin, fluproquazone,fopirtoline, fosfosal, guaimesal, guaiazolene, isonixirn, lefetamineHCl, leflunomide, lofemizole, lotifazole, lysin clonixinate,meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin,oxaceprol, oxapadol, paranyline, perisoxal, perisoxal citrate, pifoxime,piproxen, pirazolac, pirfenidone, proquazone, proxazole, thielavin B,tiflamizole, timegadine, tolectin, tolpadol, tryptamid and thosedesignated by company code number such as 480156S, AA861, AD1590,AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100,EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851,MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152,SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA60, TAI-901(4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770.Structurally related NSAIDs having similar analgesic andanti-inflammatory properties to the NSAIDs are also intended to beencompassed by this group.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatmentor concurrent treatment) with any of one or more corticosteroids,prodrug esters or pharmaceutically acceptable salts thereof for thetreatment of the diseases and disorders recited herein, including acuteand chronic inflammation such as rheumatic diseases, graft versus hostdisease and multiple sclerosis. Corticosteroids, prodrug esters andpharmaceutically acceptable salts thereof include hydrocortisone andcompounds which are derived from hydrocortisone, such as21-acetoxypregnenolone, alclomerasone, algestone, amcinonide,beclomethasone, betamethasone, betamethasone valerate, budesonide,chloroprednisone, clobetasol, clobetasol propionate, clobetasone,clobetasone butyrate, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacon, desonide, desoximerasone,dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,fluazacort, flucloronide, flumethasone, flumethasone pivalate,flucinolone acetonide, flunisolide, fluocinonide, fluorocinoloneacetonide, fluocortin butyl, fluocortolone, fluocortolone hexanoate,diflucortolone valerate, fluorometholone, fluperolone acetate,fluprednidene acetate, fluprednisolone, flurandenolide, formocortal,halcinonide, halometasone, halopredone acetate, hydro-cortamate,hydrocortisone, hydrocortisone acetate, hydro-cortisone butyrate,hydrocortisone phosphate, hydrocortisone 21-sodium succinate,hydrocortisone tebutate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodiumphosphate, prednisolone sodium succinate, prednisolone sodium21-m-sulfobenzoate, prednisolone sodium 21-stearoglycolate, prednisolonetebutate, prednisolone 21-trimethylacetate, prednisone, prednival,prednylidene, prednylidene 21-diethylaminoacetate, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide andtriamcinolone hexacetonide. Structurally related corticosteroids havingsimilar analgesic and anti-inflammatory properties are also intended tobe encompassed by this group.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more slow-acting antirheumaticdrugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS),prodrug esters, or pharmaceutically acceptable salts thereof for thetreatment of the diseases and disorders recited herein, including acuteand chronic inflammation such as rheumatic diseases, graft versus hostdisease and multiple sclerosis. SAARDs or DMARDS, prodrug esters andpharmaceutically acceptable salts thereof comprise: allocupreide sodium,auranofin, aurothioglucose, aurothioglycanide, azathioprine, brequinarsodium, bucillamine, calcium 3-aurothio-2-propanol-1-sulfonate,chlorambucil, chloroquine, clobuzarit, cuproxoline, cyclo-phosphamide,cyclosporin, dapsone, 15-deoxyspergualin, diacerein, glucosamine, goldsalts (e.g., cycloquine gold salt, gold sodium thiomalate, gold sodiumthiosulfate), hydroxychloroquine, hydroxychloroquine sulfate,hydroxyurea, kebuzone, levamisole, lobenzarit, melittin,6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil,myoral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such asSKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine.Structurally related SAARDs or DMARDs having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup.

In another specific embodiment, the present disclosure is directed tothe use of an antibody in combination (pretreatment, post-treatment, orconcurrent treatment) with any of one or more COX2 inhibitors, prodrugesters or pharmaceutically acceptable salts thereof for the treatment ofthe diseases and disorders recited herein, including acute and chronicinflammation. Examples of COX2 inhibitors, prodrug esters orpharmaceutically acceptable salts thereof include, for example,celecoxib. Structurally related COX2 inhibitors having similar analgesicand anti-inflammatory properties are also intended to be encompassed bythis group. Examples of COX-2 selective inhibitors include but notlimited to etoricoxib, valdecoxib, celecoxib, licofelone, lumiracoxib,rofecoxib, and the like.

In still another specific embodiment, the present disclosure is directedto the use of an antibody in combination (pretreatment, post-treatment,or concurrent treatment) with any of one or more antimicrobials, prodrugesters or pharmaceutically acceptable salts thereof for the treatment ofthe diseases and disorders recited herein, including acute and chronicinflammation. Antimicrobials include, for example, the broad classes ofpenicillins, cephalosporins and other beta-lactams, aminoglycosides,azoles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides,lincosamides and polymyxins. The penicillins include, but are notlimited to penicillin G, penicillin V, methicillin, nafcillin,oxacillin, cloxacillin, dicloxacillin, floxacillin, ampicillin,ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, hetacillin,cyclacillin, bacampicillin, carbenicillin, carbenicillin indanyl,ticarcillin, ticarcillin/clavulanate, azlocillin, mezlocillin,peperacillin, and mecillinam. The cephalosporins and other beta-lactamsinclude, but are not limited to cephalothin, cephapirin, cephalexin,cephradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan,cefoxitin, ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime,moxalactam, ceftizoxime, cetriaxone, cephoperazone, ceftazidime,imipenem and aztreonam. The aminoglycosides include, but are not limitedto streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycinand neomycin. The azoles include, but are not limited to fluconazole.The quinolones include, but are not limited to nalidixic acid,norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin andtemafloxacin. The macrolides include, but are not limited toerythomycin, spiramycin and azithromycin. The rifamycins include, butare not limited to rifampin. The tetracyclines include, but are notlimited to spicycline, chlortetracycline, clomocycline, demeclocycline,deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,minocycline, oxytetracycline, penimepicycline, pipacycline,rolitetracycline, sancycline, senociclin and tetracycline. Thesulfonamides include, but are not limited to sulfanilamide,sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole andco-trimoxazole (trimethoprim/sulfamethoxazole). The lincosamidesinclude, but are not limited to clindamycin and lincomycin. Thepolymyxins (polypeptides) include, but are not limited to polymyxin Band colistin.

Methods of Treatment: Pharmaceutical Formulations, Routes ofAdministration

Compositions are disclosed comprising a therapeutically effective amountof one or a plurality of the antibodies of the disclosure together witha pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative, and/or adjuvant. In addition, the disclosure providesmethods of treating a patient by administering such pharmaceuticalcomposition. A patient can be either a human subject or an animalsubject.

Pharmaceutical compositions comprising one or more antibodies can beused to reduce Factor Bb activity. Pharmaceutical compositionscomprising one or more antibodies can be used in treating theconsequences, symptoms, and/or the pathology associated with Factor Bbactivity. Pharmaceutical compositions comprising one or more antibodiescan be used in methods of inhibiting the complement pathway and/orFactor Bb binding to other complement proteins. In certain embodiments,the antibody inhibits protease activity of Factor Bb. In additionalembodiments, pharmaceutical compositions comprising one or moreantibodies can be used in methods of inhibiting Factor Bb proteaseactivity. Pharmaceutical compositions comprising one or more antibodiescan be used in methods of treating the consequences, symptoms, and/orthe pathology associated with Factor Bb activity. Pharmaceuticalcompositions comprising one or more antibodies can be used in methods ofinhibiting the production MAC. Pharmaceutical compositions comprisingone or more antibodies can be used in methods of inhibiting MacularDegeneration.

Preferably, acceptable formulation materials are nontoxic to recipientsat the dosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of Factor Bb antibodies are provided.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the disclosure. In certain embodiments, the primaryvehicle or carrier in a pharmaceutical composition can be either aqueousor non-aqueous in nature. For example, a suitable vehicle or carrier canbe water for injection, physiological saline solution or artificialcerebrospinal fluid, possibly supplemented with other materials commonin compositions for parenteral administration. Neutral buffered salineor saline mixed with serum albumin are further exemplary vehicles. Inspecific embodiments, pharmaceutical compositions comprise Tris bufferof about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and mayfurther include sorbitol or a suitable substitute therefor. In certainembodiments of the disclosure, Factor Bb antibody compositions can beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (REMINGTON'SPHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the Factor Bbantibody product can be formulated as a lyophilizate using appropriateexcipients such as sucrose.

The pharmaceutical compositions of the disclosure can be selected forparenteral delivery. Alternatively, the compositions can be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this disclosure can be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired Factor Bb antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the Factor Bb antibody is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredantibody.

Pharmaceutical compositions of the disclosure can be formulated forinhalation. In these embodiments, Factor Bb antibodies areadvantageously formulated as a dry, inhalable powder. In specificembodiments, Factor Bb antibody inhalation solutions may also beformulated with a propellant for aerosol delivery. In certainembodiments, solutions can be nebulized. Pulmonary administration andformulation methods therefore are further described in InternationalPatent Application No. PCT/US94/001875, which is incorporated byreference and describes pulmonary delivery of chemically modifiedproteins. It is also contemplated that formulations can be administeredorally. Factor Bb antibodies that are administered in this fashion canbe formulated with or without carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe Factor Bb antibody. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

A pharmaceutical composition of the disclosure is preferably provided tocomprise an effective quantity of one or a plurality of Factor Bbantibodies in a mixture with non-toxic excipients that are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or another appropriate vehicle, solutions can be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving Factor Bb antibodies insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method can beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations canbe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The disclosure alsoprovides kits for producing a single-dose administration unit. The kitsof the disclosure may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this disclosure, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of a Factor Bb antibody-containingpharmaceutical composition to be employed will depend, for example, uponthe therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication forwhich the Factor Bb antibody is being used, the route of administration,and the size (body weight, body surface or organ size) and/or condition(the age and general health) of the patient. In certain embodiments, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect. A typical dosage may range fromabout 0.1 μg/kg to up to about 30 mg/kg or more, depending on thefactors mentioned above. In specific embodiments, the dosage may rangefrom 0.1 μg/kg up to about 30 mg/kg, optionally from 1 μg/kg up to about30 mg/kg or from 10 μg/kg up to about 5 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular Factor Bb antibody in the formulation used. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data. In certain embodiments, the antibodiesof the disclosure can be administered to patients throughout an extendedtime period. Chronic administration of an antibody of the disclosureminimizes the adverse immune or allergic response commonly associatedwith antibodies that are not fully human, for example an antibody raisedagainst a human antigen in a non-human animal, for example, a non-fullyhuman antibody or non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intravitreal,sub-retinal, intraarterial, intraportal, or intralesional routes; bysustained release systems or by implantation devices. In certainembodiments, the compositions can be administered by bolus injection orcontinuously by infusion, or by implantation device.

The composition also can be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device can be implanted intoany suitable tissue or organ, and delivery of the desired molecule canbe via diffusion, timed-release bolus, or continuous administration. Forocular implants, the implant can be implanted via intra-ocularinjection, intravitreal injection, sub-retinal injection, suprachoroidalinjection, retrobulbar injection or injection into sub-Tenon space.

It also can be desirable to use Factor Bb antibody pharmaceuticalcompositions according to the disclosure ex vivo. In such instances,cells, tissues or organs that have been removed from the patient areexposed to Factor Bb antibody pharmaceutical compositions after whichthe cells, tissues and/or organs are subsequently implanted back intothe patient.

In particular, Factor Bb antibodies can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the Factor Bbantibody. In certain embodiments, such cells can be animal or humancells, and can be autologous, heterologous, or xenogeneic. In certainembodiments, the cells can be immortalized. In other embodiments, inorder to decrease the chance of an immunological response, the cells canbe encapsulated to avoid infiltration of surrounding tissues. In furtherembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

All references cited within the body of the instant specification arehereby expressly incorporated by reference in their entirety.

EXAMPLES

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the disclosure.

Example 1—Binding Assay of Anti-Factor Bb Antibody Compared toAnti-Factor B Antibody

Bio-Layer Interferometry (BLI), a label-free technology was used formeasuring the binding kinetics of Factor Bb (CompTech®) and human FactorB antigen (CompTech®) with anti-Factor Bb monoclonal antibody. Affinitymeasurements were performed with Octet QK^(e) equipped with Anti-humanIgG Fc capture (AHC) biosensor tips (FortéBio®, Menlo Park, Calif.,USA). The assay was performed at 30° C. in 1×PBS buffer (Gibco®, PBSpH7.2). Samples were agitated at 1000 rpm. Prior to analysis, sensorswere humidified for 15 minutes.

Purified anti-Factor Bb antibody was tested for its binding capacitywith AHC sensor tips. Tips were loaded using 20 μg/ml of anti-Factor Bbantibody. Loading proceeded for 300 s resulting in capture levels ofbetween 1.8 and 2 nm. Factor Bb or Factor B antigens were prepared forbinding analysis by dilution to concentrations of 50 nM in 1×PBS.Association was initiated and monitored for 200 s, after which tips weretransferred to 1×PBS buffer without Factor protein (Gibco, PBS pH 7.2),in order to monitor dissociation. Sensor data was collected throughoutthe experiments, processed, and analyzed using the Octet data analysissoftware 7 (Forte Bio).

Selectivity of anti-factor Bb antibody was first tested by comparingbinding on-rates for Factor Bb and Factor B proteins. This analysis wasperformed using the Octet QK^(e) system from Forte Bio®. The binding,measured over 200 s in protein preparations of 50 nM, indicate that theanti-factor Bb antibody specifically binds to Factor Bb, but binding toFactor B is significantly lower (FIG. 1).

Example 2—Functional Assay of Anti-Factor Bb Monoclonal Antibody

Hemolysis Assay—

Activation of the alternative pathway of (AP) requires higherconcentrations of serum than the classical pathway. Generally, a finalconcentration of 5 mM Mg++ in the presence of 5 mM EGTA is used in theassays where the EGTA chelates Ca⁺⁺ preferentially. The AP of mostmammalian species is activated spontaneously by rabbit erythrocytes sothey are a convenient target. Prepare rabbit erythrocytes (ComplementTechnology, Inc.) by washing 3 times with GVB0 (CompTech product) andresuspending into 5×10⁸/ml. Different amount of anti-factor Bb antibodywas diluted with GVB0. Mix the 100 ul reaction on ice in the order ofserial diluted anti-factor Bb antibody, 0.1M MgEGTA (CompTech product),1/2NHS (normal human serum diluted 1/2 with GVB0), and rabbit Er. Then,incubate the reaction at 37° C. for 30 minutes on a shaker. Add 1.0 mlcold GVBE. Mix and centrifuge for 3 min at approx. 1000×g, or higher, topellet cells. Transfer 100 ul of the supernatant to a 96-well plate andread at 412 nm (SoftMax Pro 4.7.1). Data was analysized using GraphPadPrism 4.

Results—

To determine the potency of anti-factor Bb antibodies, AP hemolysisassay was performed and the IC50 nM (the amount of the antibodynecessary to inhibit 50% of the hemolysis reaction). The data indicatedthat the IC50 of the presently disclosed anti-Factor Bb antibody isabout 40 nM, whereas IC50 of anti-Factor B antibody is about 100 nM(FIG. 2). Thus, anti-Factor Bb antibody is about ten times more potentthan anti-Factor B antibody in AP hemolysis assay.

Example 3—In Vivo Efficacy Model

Humanized H4L4 99A12 antibody, (SEQ ID NOS:15 and 11 respectively), wastested in a non-human primate light injury model. Intravitreal dosing ofthe H4L4 99A12 antibody provided efficacy in blocking complementdeposition in the retina relative to control. This data indicates thatlocal delivery of the H4L4 99A12 antibody is efficacious in an in vivomodel relevant to treatment in humans of macular degeneration and otherocular indications.

1. (canceled)
 2. A nucleic acid encoding an isolated antibody, whereinthe antibody is a Factor Bb antibody, wherein the antibody binds toFactor Bb with greater affinity than to Factor B; and inhibitscomplement dependent hemolysis.
 3. The nucleic acid of claim 2, whereinthe antibody binds Factor Bb with a K_(d) of less than about 1 nM. 4.The nucleic acid of claim 2, wherein the antibody blocks formation ofmembrane attack complex (MAC) in a patient.
 5. The nucleic acid of claim2, comprising a heavy chain and a light chain.
 6. The nucleic acid ofclaim 5, wherein the light chain comprises an amino acid sequence atleast 80% identical to a sequence selected from the group consisting ofSEQ ID NO: 8-11; and wherein the heavy chain comprises an amino acidsequence of at least 80% identical to a sequence selected from the groupconsisting of SEQ ID NO: 12-15.
 7. The nucleic acid of claim 5, whereinthe light chain comprises an amino acid sequence at least 80% identicalto a sequence selected from the group consisting of SEQ ID NO: 24-27;and wherein the heavy chain comprises an amino acid sequence at least80% identical to a sequence selected from the group consisting of SEQ IDNO: 28-31.
 8. The nucleic acid of claim 5, wherein the antibodycomprises a light chain variable domain and a heavy chain variabledomain selected from the light and heavy chain variable domain aminoacid sequences: SEQ ID NO:8/SEQ ID NO:12; SEQ ID NO:8/SEQ ID NO:13; SEQID NO:8/SEQ ID NO:14; SEQ ID NO:8/SEQ ID NO:15; SEQ ID NO:9/SEQ IDNO:12; SEQ ID NO:9/SEQ ID NO:13; SEQ ID NO:9/SEQ ID NO:14; SEQ IDNO:9/SEQ ID NO:15; SEQ ID NO:10/SEQ ID NO:12; SEQ ID NO:10/SEQ ID NO:13;SEQ ID NO:10/SEQ ID NO:14; and SEQ ID NO:10/SEQ ID NO:15; SEQ IDNO:11/SEQ ID NO:12; SEQ ID NO:11/SEQ ID NO:13; SEQ ID NO:11/SEQ IDNO:14; and SEQ ID NO:11/SEQ ID NO:15.
 9. The nucleic acid of claim 5,wherein the antibody comprises a light chain variable domain and a heavychain variable domain selected from the light chain and heavy chainvariable domain amino acid sequences: SEQ ID NO:24/SEQ ID NO:28; SEQ IDNO:24/SEQ ID NO:29; SEQ ID NO:24/SEQ ID NO:30; SEQ ID NO:24/SEQ IDNO:31; SEQ ID NO:25/SEQ ID NO:28; SEQ ID NO:25/SEQ ID NO:29; SEQ IDNO:25/SEQ ID NO:30; SEQ ID NO:25/SEQ ID NO:31; SEQ ID NO:26/SEQ IDNO:28; SEQ ID NO:26/SEQ ID NO:29; SEQ ID NO:26/SEQ ID NO:30; and SEQ IDNO:26/SEQ ID NO:31; SEQ ID NO:27/SEQ ID NO:28; SEQ ID NO:27/SEQ IDNO:29; SEQ ID NO:27/SEQ ID NO:30; and SEQ ID NO:27/SEQ ID NO:31.
 10. Thenucleic acid of claim 5, wherein the antibody comprises a light chainvariable domain amino acid sequence of SEQ ID NO:11 and a heavy chainvariable domain amino acid sequence of SEQ ID NO:15.
 11. The nucleicacid of claim 2, wherein said antibody is a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a humanized antibody, achimeric antibody, a multispecific antibody, or an antibody fragmentthereof.
 12. The nucleic acid of claim 11, wherein said antibodyfragment is a Fab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fvfragment, a diabody, or a single chain antibody molecule.
 13. Thenucleic acid of claim 11, wherein said antibody is of the IgG1-, IgG2-IgG3- or IgG4-type.